CN106178162B - Treating AIDS organelle - Google Patents
Treating AIDS organelle Download PDFInfo
- Publication number
- CN106178162B CN106178162B CN201610540997.9A CN201610540997A CN106178162B CN 106178162 B CN106178162 B CN 106178162B CN 201610540997 A CN201610540997 A CN 201610540997A CN 106178162 B CN106178162 B CN 106178162B
- Authority
- CN
- China
- Prior art keywords
- cell
- organelle
- hiv
- blood
- macrophage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000003463 organelle Anatomy 0.000 title claims abstract description 64
- 208000030507 AIDS Diseases 0.000 title claims abstract description 61
- 210000004027 cell Anatomy 0.000 claims abstract description 162
- 210000002381 plasma Anatomy 0.000 claims abstract description 69
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000011282 treatment Methods 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 13
- 239000000463 material Substances 0.000 claims abstract description 10
- 108090000695 Cytokines Proteins 0.000 claims abstract description 6
- 102000004127 Cytokines Human genes 0.000 claims abstract description 6
- 239000012634 fragment Substances 0.000 claims abstract description 4
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims abstract 5
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims abstract 5
- 210000004369 blood Anatomy 0.000 claims description 46
- 239000008280 blood Substances 0.000 claims description 46
- 239000007788 liquid Substances 0.000 claims description 28
- 230000004087 circulation Effects 0.000 claims description 14
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 13
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 13
- 230000001464 adherent effect Effects 0.000 claims description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 201000010099 disease Diseases 0.000 claims description 10
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 4
- 239000000835 fiber Substances 0.000 claims description 4
- 238000000684 flow cytometry Methods 0.000 claims description 4
- 229960002897 heparin Drugs 0.000 claims description 4
- 229920000669 heparin Polymers 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 239000000470 constituent Substances 0.000 claims 2
- 230000005298 paramagnetic effect Effects 0.000 claims 2
- 229920002472 Starch Polymers 0.000 claims 1
- 210000001776 amniocyte Anatomy 0.000 claims 1
- 235000019698 starch Nutrition 0.000 claims 1
- 210000002540 macrophage Anatomy 0.000 abstract description 90
- 230000000694 effects Effects 0.000 abstract description 18
- 239000011324 bead Substances 0.000 abstract description 12
- 230000012010 growth Effects 0.000 abstract description 11
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000001727 in vivo Methods 0.000 abstract description 7
- 238000004321 preservation Methods 0.000 abstract description 6
- 230000003321 amplification Effects 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 230000002924 anti-infective effect Effects 0.000 abstract description 3
- 230000000242 pagocytic effect Effects 0.000 abstract description 3
- 230000000717 retained effect Effects 0.000 abstract 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 68
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 18
- 238000001514 detection method Methods 0.000 description 17
- 239000006285 cell suspension Substances 0.000 description 14
- 206010057249 Phagocytosis Diseases 0.000 description 13
- 230000008782 phagocytosis Effects 0.000 description 13
- 238000002360 preparation method Methods 0.000 description 13
- 210000000952 spleen Anatomy 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 12
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 210000001616 monocyte Anatomy 0.000 description 11
- 210000001744 T-lymphocyte Anatomy 0.000 description 10
- 239000012980 RPMI-1640 medium Substances 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000012544 monitoring process Methods 0.000 description 7
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 102100034349 Integrase Human genes 0.000 description 6
- 210000004970 cd4 cell Anatomy 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 210000001539 phagocyte Anatomy 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000003462 vein Anatomy 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 101710205625 Capsid protein p24 Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 101710177166 Phosphoprotein Proteins 0.000 description 5
- 101710149279 Small delta antigen Proteins 0.000 description 5
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 5
- 102100022563 Tubulin polymerization-promoting protein Human genes 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 210000005259 peripheral blood Anatomy 0.000 description 5
- 239000011886 peripheral blood Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000004062 sedimentation Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 208000031886 HIV Infections Diseases 0.000 description 4
- 208000037357 HIV infectious disease Diseases 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000017531 blood circulation Effects 0.000 description 4
- 210000001772 blood platelet Anatomy 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000005138 cryopreservation Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 108010041397 CD4 Antigens Proteins 0.000 description 3
- 101710091045 Envelope protein Proteins 0.000 description 3
- 101710188315 Protein X Proteins 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 3
- 101710090322 Truncated surface protein Proteins 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 210000004381 amniotic fluid Anatomy 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 230000023555 blood coagulation Effects 0.000 description 3
- 210000000234 capsid Anatomy 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 210000003714 granulocyte Anatomy 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- 210000004493 neutrocyte Anatomy 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 208000031957 HIV carrier Diseases 0.000 description 2
- 241000167880 Hirundinidae Species 0.000 description 2
- 101000950847 Homo sapiens Macrophage migration inhibitory factor Proteins 0.000 description 2
- 108010061833 Integrases Proteins 0.000 description 2
- 108010070716 Intercellular Signaling Peptides and Proteins Proteins 0.000 description 2
- 102000005755 Intercellular Signaling Peptides and Proteins Human genes 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229920006221 acetate fiber Polymers 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000001631 haemodialysis Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000003595 mist Substances 0.000 description 2
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000004745 nonwoven fabric Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000035485 pulse pressure Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000009256 replacement therapy Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009747 swallowing Effects 0.000 description 2
- 239000000052 vinegar Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010001526 Air embolism Diseases 0.000 description 1
- 235000010894 Artemisia argyi Nutrition 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000015612 Complement 3b Receptors Human genes 0.000 description 1
- 108010024114 Complement 3b Receptors Proteins 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- 102100022133 Complement C3 Human genes 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 101710114816 Gene 41 protein Proteins 0.000 description 1
- 229940099797 HIV integrase inhibitor Drugs 0.000 description 1
- 229940122440 HIV protease inhibitor Drugs 0.000 description 1
- 206010059484 Haemodilution Diseases 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- 241000668842 Lepidosaphes gloverii Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920002560 Polyethylene Glycol 3000 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 239000006096 absorbing agent Substances 0.000 description 1
- UELITFHSCLAHKR-UHFFFAOYSA-N acibenzolar-S-methyl Chemical compound CSC(=O)C1=CC=CC2=C1SN=N2 UELITFHSCLAHKR-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000027645 antigenic variation Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 244000030166 artemisia Species 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008081 blood perfusion Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 201000003373 familial cold autoinflammatory syndrome 3 Diseases 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 239000003084 hiv integrase inhibitor Substances 0.000 description 1
- 239000004030 hiv protease inhibitor Substances 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000034217 membrane fusion Effects 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 210000001711 oxyntic cell Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3692—Washing or rinsing blood or blood constituents
Landscapes
- Health & Medical Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Anesthesiology (AREA)
- Cardiology (AREA)
- Hematology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to the treating AIDS organelles of field of biomedicine, it is characterized in that isolating CD14 cell with immunomagnetic beads method, and then it prepares and had not only retained former macrophage feature but also the hybridoma macrophage strain of energy indeterminate growth, it selects to make amplification cultivation and preservation to the strong phagocytic clone of HIV, thus the hybridoma macrophage strain prepared prepared with high-biocompatibility material package can prevent cell and its fragment even HIV from filtering out, HIV capable of being swallowed for cell strain, the organelle in place is provided, extracorporal circulatory system is constituted with plasma separator, isolated blood plasma is after organelle filters out, HIV therein is swallowed by macrophage strain to be removed, the anti-infective macrophage cytokines generated simultaneously feed back internal with blood plasma, the AIDS hybridoma technology of macrophage cytokines needed for not only having removed HIV to realization but also supplemented is external New treatment, than being confined to kill HIV in vivo but the traditional remedies that are difficult to realize are more feasible and have no toxic side effect.
Description
Technical field
The present invention relates to treating AIDS organelles in field of biomedicine, and it is sensitive mainly to prepare HIV by artificial industry
Cell achievees the purpose that treat AIDS for swallowing the free HIV in blood plasma in vitro.
Background technique
AIDS is passed caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV)
It catches an illness, is widely current in the whole world, according to the related report of the World Health Organization (WHO) and The Joint Programme on AIDS, certainly
Since U.S.'s discovery Patient With Aids cases in 1981, the whole world has 208 countries and regions to receive the serious of AIDS so far
It threatens, there are about 40,000,000 people to have infected AIDS, and death toll is more than 20,000,000, and there are about 6000 people to become AIDS sense daily
Dye person, while there are about more than 300 people to die of AIDS daily.It is in rapid growth period in HIV infected individuals in China, it is far super at present
Cross 1,000,000.AIDS has become the great of after tumour, cardiovascular and cerebrovascular disease, tuberculosis, diabetes another facing mankind
Infectious diseases, it has also become the serious public health of global concern and social concern.
HIV is the retrovirus for infecting human immune cells, and about 120 nanometers of diameter, substantially spherical in shape, outer membrane is lipoid
Coating (comes from host cell), and embedding virulent albumen gp120 and gp41, gp41 are transmembrane proteins, and gp120 is located at surface,
And with gp41 by noncovalent interaction in conjunction with, be inward from by the albumen p17 sphere matrix (Matrix) formed and albumen p24
The half-cone capsid (Capsid) of formation, capsid include virulent rna gene group, enzyme (reverse transcriptase, integrase, protease)
And other ingredients from host cell.
After HIV enters human body, first by the phagocytosis of macrophage, but HIV changes certain portions in macrophage quickly
The acidic environment of position creates the condition for being suitble to it to survive, and is not killed mass propagation aggregation in it instead not only.Because
CD4 is the receptor of HIV, so the HIV bred in macrophage is by its envelope protein gp120 and under the auxiliary of gp41
It is (thin that (gp41 plays a part of bridge, utilizes itself hydrophobic effect mediate retroviral cyst membrane and cell membrane fusion) enters CD4+ cell
Born of the same parents, mononuclear macrophage, Dendritic Cells etc.), it is proliferated rapidly in the cell, generates 10 daily9~1010Virion, not
Disconnected other normal and regenerated CD4+ that enter are replicated into the cell, are manufactured more virus infected cells, are kept peripheral blood CD4+T thin
Born of the same parents' sustaining breakdown is reduced.CD4+T cell is most important immunocyte, and the infected once loses a large amount of CD4+T cells, whole
A immune system will all lose resistance to the infection of various diseases by deathblow；HIV enters host's CD4+ cell
It can also show as hiding without showing clinical symptoms for a long time afterwards, geneome RNA reverse transcription is whole with virus at double-stranded DNA
Synthase enters in host cell nuclear, and under the action of integrase, double-stranded DNA is integrated into host cell gene group, is integrated
Viral DNA is known as provirus, and the several months that can hide does not replicate even for many years, causes the incubation period of AIDS several months to many years.In AIDS
Incubation period, HIV mainly breeds in the macrophage of lymph node and Dendritic Cells, these cells are intracorporal HIV storages
Library, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, mitogen, antigen, TNF, IL-2 and lymph element
(LT) HIV provirus gene can be excited to replicate in the CD4+T Intracellular transcription of infection.After being largely proliferated, inhibition of HIV particle
Blood is constantly discharged and be free on from the infection cell being destroyed, and is then entered back into other cells and is continued course of infection.
Body production envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody can be stimulated after HIV infection.?
Low-level antiviral neutralizing antibody is measured in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum,
HIV carriers' highest illustrates that the antibody has protective effect in vivo.But antibody cannot be with the virus that retains in mononuclear macrophage
Contact, and antigenic variation easily occurs for HIV envelope protein, original antibody is ineffective, keeps neutralizing antibody due from playing
Effect.In the latent infection stage, HIV provirus is integrated into host cell gene group, therefore HIV will not be known by immune system
Not, so relying solely on itself immune function can not be removed.Another critically important reason is to be killed, removed according to antibody
After the mechanism of antigen, immune antibody and antigen binding, to generate immunological effect or by activating complement, mediate ADCC effect
Cellular antigen should be dissolved, but HIV is not cellular antigen；Phagocyte phagocytosis is attracted to remove by chemotaxis anti-
Original, but HIV is protected in phagocyte instead, is proliferated；Antibody and antigen binding play neutralization, make to lose infection
Power, but HIV can not be killed by immune system, be removed.
The blood purification technology that 20th century began one's study refers to outside the blood lead body patient and is filled by a kind of purification
Set, remove some of them morbid substance, purify blood, then feed back into vivo, achieve the purpose that treat disease.The blood of early stage is net
Change and often refer to hemodialysis technology, uremic patient kidney replacement therapy is used for, with the progress of medicine and the hair of apparatus for purifying blood
Exhibition, derives different blood purification modes, such as plasma exchange, blood perfusion, immuno absorbence, blood on the basis of haemodialysis
Liquid filtration etc..The application of different blood purification technologies substantially improves the treatment of certain difficult complex diseases, mainly applies blood plasma
Replacement technique separates and removes macromolecular immune complex class virulence factor to mitigate, adjust and treat certain immunity diseases.
Since the 1980s, the First Academy of Zhejiang University Li Lan Juan team starts to achieve good therapeutic effect with replacement therapy of blood plasma hepatic failure,
And abiotic type, bion, hybrid artificial liver support system and the dialysis of related artificial liver have been founded on this basis
The improvement and innovation of the artificial liver system unit such as device, transfusion filters, active carbon (resin) absorber, bioreactor.
But it has no and HIV infection cell and the report of free HIV is removed with blood purification technology.
Commonly antiviral first-line drug is reverse transcriptase inhibitor to treating AIDS at present, which cannot kill HIV, deposit
Individual difference, drug resistance, gland plastochondria toxicity, bone marrow suppression, globular anemia, granulocyte and decrease of platelet, pancreatitis,
A variety of toxic side effects such as hepatic injury, other as hiv protease inhibitor, hiv integrase inhibitor, cell factor, vaccine therapy,
Gene therapy and monoclonal antibody passive immunization therapy etc. are difficult to because of a variety of causes commonly used.There is document report sharp
The stimulant for using the monoclonal antibody of T cell surface C D3 molecule to grow as cell, the T of mass propgation AIDS patient separation
It after cell, is fed back as itself therapeutic cells, but HIV is bred also with the culture of HIV infection cell in endogenous multiplication, is increased
The feedback of amount T cell also results in the feedback of increment HIV.
The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophil leucocyte in peripheral blood, macrophagocyte
It is the macrophage in the monocyte and a variety of organs, tissue in blood, the two constitutes mononuclear phagocyte system.Monocyte
It is formed by the monocyte precursor Development And Differentiation in marrow, accounts for about 3% one the 8% of blood middle leukocytes sum, volume is relatively drenched
Bar cell is bigger, and monocyte only stops 12-24 hours in blood, subsequently into connective tissue or organ, reach maturity for
Macrophage, macrophage are highly differentiation, mature cell type in mononuclear phagocyte system, have stronger phagocytosis function
Can, wandering macrophage is greater than monocyte several times, and it lasts a long time, can survive in the tissue some months, the macrophage of colonization
There is different titles, be Kupffer Cell in liver, be in brain microglia, be osteoclast etc. in bone, expresses Fc
Receptor, C3b receptor and CDl4 play defense function in inherent immunity, and the professional antigen of participation adaptive immunity is offered
Cell.The CD4 molecule of Expression of Macrophages, is the receptor of AIDS virus (HIV), thin by macrophage first after HIV enters human body
The phagocytosis of born of the same parents, but HIV changes the acidic environment at certain positions in macrophage quickly, creates the condition for being suitble to it to survive,
It is not killed mass propagation aggregation in it instead not only, then HIV is passed into CD4+T cell.
In conclusion the CD4 molecule of Expression of Macrophages, is the receptor of AIDS virus (HIV), it is first after HIV enters human body
First by the phagocytosis of macrophage, but HIV changes the acidic environment at certain positions in macrophage quickly, creates and is suitble to it
The condition of existence is not killed mass propagation aggregation in it instead not only, then HIV is passed to CD4+T cell, last dead
In the various diseases that CD4+T cell considerable damage, function are lost and cause.
Human body lacks the immunity for killing HIV, and the curative effect of medication used in vivo is bad and toxic side effect is big, and AIDS is long
Attack the global problem being unable to.
Summary of the invention
In order to solve to attack the global problem in the treating AIDS field being unable to long, present inventors have proposed the present invention.
The invention aims to provide treating AIDS organelle；Another object is to provide for the preparation of organelle and answers
Use method.
The object of the present invention is achieved like this: isolating CDl4 cell with immunomagnetic beads method, and then prepares and both retain original
The hybridoma macrophage strain of macrophage feature and energy indeterminate growth selects to make amplification cultivation to the strong phagocytic clone of HIV
And preservation, by the hybridoma macrophage strain thus prepared with high-biocompatibility material package and prepare can prevent cell and its
Fragment even HIV filter out, can be cell strain phagocytosis HIV the organelle in place be provided, with plasma separator composition extracorporal circulatory system,
Isolated blood plasma is after organelle filters out, and HIV therein is swallowed by macrophage strain to be removed, while the anti-infective macrophage generated is thin
Intracellular cytokine is fed back in vivo with blood plasma, to realize the AIDS hybridoma skill not only removed HIV but also supplemented required macrophage cytokines
The external new treatment of art, than being confined to kill HIV in vivo but the traditional remedies that are difficult to realize are more feasible and have no toxic side effect.
The present invention separates macrophage with classical magnetic activated cell seperation, thin with classical hybridoma technology preparation macrophage
Born of the same parents' hybridoma cell strain is allowed to not only retain the activity of phagocytosis HIV, but also myeloma cell's characteristic with indeterminate growth, through expanding
Treating AIDS organelle is made with high-biocompatibility material package afterwards, the CD4 molecule of Expression of Macrophages therein is Chinese mugwort
The receptor of virus to be grown, there is the characteristic of natural phagocytosis HIV, the present invention simulates non-specific phagocytosis mode in macrophage body, when
When blood plasma containing HIV flows through organelle, HIV is in contact with macrophage and is swallowed, and the blood plasma and blood after removing HIV are thin
Born of the same parents feed back after converging, and internal HIV can be made constantly to be reduced by removing in the treatment until disappearing, can effectively block newborn CD4+
T cell is again by HIV infection, to reach the therapeutic purposes of immunologic reconstitution, and is difficult to kill HIV in vivo at present and be difficult to effective
It blocks again to the infection cycle approach of CD4+T cell from blood plasma to macrophage, and somewhat expensive, the big routine of medicine side effect are anti-
Reverse transcription treatment is compared, and the present invention is more economical, almost without medicine side effect, realize manually by HIV from be transferred in vivo it is external and
The treatment new method of removing.
Specific embodiment
Fig. 1 is the application schematic diagram of the treating AIDS organelle proposed according to the present invention.
Fig. 2 is the internal structure chart of the plasma separator proposed according to the present invention.
Fig. 3 is the internal structure chart of the treating AIDS organelle proposed according to the present invention.
In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump
(3) it is connected with plasma separator (4), plasma separator (4) cell in parallel with two through blood plasma pump (6) and circulation line (7)
Device (8), organelle (9) be connected, be then successively connected with circulation line (10), venous line (5), venous line (5) it is another
End is connected with vein blood vessel.
In Fig. 2,1 is plasma separator, and 2 be plasma separator inner cavity, and 3 be the micropore on the tube wall of plasma separator inner cavity, 4
Being cannot be by the haemocyte of micropore (3), and 5 be the small molecule blood plasma components that can pass through micropore (3), and 6 be plasma separator exocoel,
7 be blood plasma outflux, and 8 be switchable valve.
In Fig. 3,1 is organelle, and 2 be hybridoma macrophage strain, and 3 be the free HIV into organelle, 4 be HIV with
The conjugate of hybridoma macrophage strain, 5 be the cell factor that hybridoma macrophage strain generates.
Below with reference to Fig. 1, Fig. 2 and Fig. 3, the embodiment for the treatment of AIDS organelle proposed by the present invention is made detailed
Description.
1, primary cell source
(1) single core blood cell: refer to the lymphocyte separated from blood with density-gradient centrifugation method and monokaryon macrophage
Cell.Specific method is: the Cord blood buying the White Blood Cells Concentrate of Blood Center or saving for scientific research takes 2mL sample, PBS
6mL anticoagulation is slowly superimposed on dropper that 4mL lymph has been added is thin by 2~3 times of hemodilution, after mixing well by liquid along tube wall
Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tube of born of the same parents' separating liquid；It is divided into pipe after centrifugation
3 layers, upper layer is blood plasma and PBS liquid, and lower layer is mainly red blood cell and granulocyte, and middle layer is lymphocyte separation medium, in upper, middle layer
It is PBMC that, which there be the white cloud and mist layer narrow band based on mononuclearcell in interface, is inserted into cloud and mist layer with capillary syring, is drawn
PBMC is placed in another 50mL centrifuge tube, is added 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant
Cell is resuspended in 50mLPBS, is centrifuged (350r/min, 20 DEG C) 15min, abandons supernatant, and Buffer (PBS+0.5% new life ox blood is added
+ 2mmol/LEDTA clearly, pH7.2) 2mL resuspension cell, it takes 15uL cell suspension to be added on blood counting chamber and counts 4 under microscope
Cell (PBMC) sum in a block plaid.
(2) it single core histocyte: is provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen
Macrophage, preparation method are: the 1. acquisition and transhipment of spleen tissue: in the approval of the reason committee and patient's informed consent
Under, the spleen sample tissue for taking operation to cut off shreds into volume about 1mm immediately3Small tissue blocks, move into equipped with pre-cooling 4 DEG C
In sterile sealing bottle, it is transported to cell culture chamber rapidly.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to
Aseptic operating platform, PBS are washed 3 times, and RPMI-1640 is washed 2 times, to remove the blood in tissue and guarantee the sterile of tissue.Machine
Tool grinds spleen tissue, at this moment just has a large amount of histocyte is outstanding to be mixed in RPMI-1640 liquid.With 200 mesh stainless steel filtering net mistakes
Filter is outstanding to be mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing red blood cell, lymphocyte,
Macrophage etc.).3. the cracking of red blood cell in spleen tissue cell suspension: and then centrifugation is washed with RPMI-1640 liquid
(1000r/min, 3min) is added Tris-NH4Cl and acts on 5min, splitting erythrocyte, Quick spin to remove cell debris
(1000r/min, 3min), remove supernatant in splitting erythrocyte fragment, PBS washing centrifugation 3 times, RPMI-1640 wash from
The heart 1 time, to remove Tris-NH4Cl remaining in suspension, it is avoided to influence the survival of cell, at this point, mainly containing in suspension
Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as culture
Cell stoste, Trypan Blue determine vigor and count, and are (3~5) × 10 with RPMI-1640 liquid adjustment cell concentration6/ L, will
The cell suspension inoculation of concentration is adjusted in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO2, 100% humidity, point
Not Pei Yang 2~3h, observe form under phase contrast microscope.The digestion of adherent spleen tissue macrophage: adherent spleen tissue macrophage
The digestion of cell: sucking culture supernatant, and macrophage is adherent, and PBS blows and beats repeatedly, digests, the washing centrifugation of gained cell suspension
(1000r/min, 3min), the macrophage isolated and purified.Further, it is also possible to which the sample discarded after treatment or operation is taken to mention
Take preparation, such as cavum peritoneale liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa.
(3) amniotic fluid, villus cell: Zhejiang University's attached hospital for obstetrics and gynaecology's reproduction heredity laboratory is spare.In reason committee member
Can ratify under patient's informed consent, take laboratory diagnosis report after remaining amniotic fluid, villus cell, select logarithmic growth phase cell
Continue to employ.
2, cell culture and the adherent preliminary sorting of macrophage
Routinely cell culture, but according to the difference of cellularity, appropriate adjustment incubation time, condition of culture etc., generally
Single core blood cell (PBMC) or single core histocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method
Culture dish in, in 37 DEG C, 5%CO2Cell incubator (Themo electro corporation CLASS 100, beauty
State) in be incubated for 2h, after mononuclearcell is adherent, inhale abandon upper layer suspension cell (cell other than macrophage be not easy it is adherent and
Removed with upper liquid), PBs buffer gently washs 3 times, and a small amount of mono- 1640 culture medium of RPMI is added, scrapes patch with cell scraper
Parietal cell (predominantly macrophage, but there are also other a small amount of attached cells).1000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid is thin
There is cell growth clone in born of the same parents, villus cell culture 1~7 day, cell growth converges the logarithmic growth phase that rate reaches 60~80%
Cell is digested with pancreatin, and PBS cleaning obtains cell suspension, is made into proper cell concentration.
3, cd4 cell sorts
Sort cd4 cell: 1. main agents and instrument using immunomagnetic beads method: (German U.S.A day Ni is biological for CD4 immunomagnetic beads
Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；Newborn bovine serum
(Hyclone company)；MiniMACS magnetic separation system (German Mei Tian Ni Bioisystech Co., Ltd).2. cd4 cell is immune
Magnetic bead sorting method: cell suspension, which is divided equally to two 1.5mLEppendorf, manages, and is centrifuged (300r/min, 20 DEG C) 10min, discards
Supernatant is resuspended the every 80uLBuffer of cell and contains cell number 107It is a, every 107A cell add 20uLCD4MicroBeads or
CD8MicroBeads is mixed well, and in 4~8 DEG C of hatching 15min, washs cell with 1mLBuffer, be centrifuged (300r/min, 20
DEG C) 10min, it discards supernatant 500uLBuffer and cell is resuspended, MS splitter is placed in the magnetic field of MACS separator, with
500uLBuffer rinsing is rinsed splitter repetitive operation 3 times by 500uL cell suspension by splitter with 500uLBuffer,
Efflux is collected, contains non-cd4 cell in efflux, splitter is taken out from separator, with 1000uLBuffer pressure flush point
From column, collection efflux, for cd4 cell, (cell viability detection: taking 15uL cell suspension respectively before and after cell purification and waits bodies for this
Product trypan blue solution mixing, the not colored shinny person of microscopically observation are living cells, and the coloring person of swelling is dead cell, calculate 200
The percentage of living cells in a cell).The cell sorted at this time is mainly macrophage.
4, CDl4 cell (macrophage) sorts
CDl4 is monocyte and the distinctive surface marker of macrophage, theoretically if from single core histocyte, sheep
It is sorted in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, gained
Cell includes monocyte and macrophage；But because the monocyte service life is short, only survive 1 day in peripheral blood and can not show a candle to macrophage
Cell is easy to adherent growth, so removing substantially in cell adhere-wall culture of the invention, the cell sorted out is essentially
Macrophage.
Basic skills is analogous to cd4 cell, using immunomagnetic beads method.1. reagent: people's CDl4 immunomagnetic beads kit
(Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead and spy
Anisotropic one monocyte of target cell combines: every 1 × 108The magnetic bead and 800uL buffering of 200uL coupling CDl4 antibody is added in a PBMC
Liquid (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/L EDTA0.5mL, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube
It mixes well, 4 DEG C of incubation 15min, centre can slightly shake 1 time.Take out centrifuge tube after 15min, every 1 × 107A cell is added 1
Buffer is pre-chilled in~2mL, and 1000r/min is centrifuged 8min, abandons supernatant, and 0.5mL buffer is added and blows and beats into single cell suspension.
(B) it collects the monocyte of marked by magnetic bead: cell splitter is placed on MACS magnetic frame, 1mL buffer statocyte is added
Splitter drips to no liquid, immediately adds above-mentioned cell suspension in people's cell splitter, rinses cell with 0.5mL buffer
Splitter 3 times.After to be rinsed, 1mL buffer is added, the emigrated cells splitter from magnetic frame is quickly pushed with needle column,
Go out the cell combined in splitter with one magnetic bead of CDl4 antibody, the as macrophage of CDl4+.
In addition, following 2 kinds of methods sorting, including 1. adherent method also can be used: PBMC being placed in and is cultivated containing RPMI-1640
In the culture dish of base, in 37 DEG C, contain 5%CO: cell incubator (Themo electro corporation CLASS 100,
The U.S.) in be incubated for 2h.It after adherent mononuclear cells, inhales and abandons upper layer suspension cell, PBs buffer gently washs 3 times, is added a small amount of
Mono- 1640 culture medium of RPMI, scrapes attached cell with cell scraper.1000r/min is centrifuged 5min, abandons supernatant.2. flow cytometry
Method: CDl4 label: taking PBMC, is adjusted with buffer (bovine serum albumin(BSA) 2.5mL and the 2mol/LEDTA 0.5mL containing 10%)
Whole cell density is 1 × 108CDl4+-FITC antibody 100uL is added in/mL in every milliliter of cell suspension, and 4 DEG C are protected from light label
18min, then 1mL streaming buffer is added to terminate dyeing into centrifuge tube, PBs is washed 3 times, with the PBS for containing 2% mycillin
Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation in flow cytometer (BD FAcsAria
II, the U.S.) on sort, according to the fluorescence intensity of CDl4 antibody, the relative particle of the relative size of cell and cell and interior
The complexity of portion's structure collects the cell of CDl4+.
5, prepared by CDl4 hybridoma cell strain (strain of hybridoma macrophage)
(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire ox
Serum (FBS) purchases Jinshi City on daytime ocean Hao biological products science and technology responsibility Co., Ltd；DMSO (-- methyl sulfoxide) it is that domestic analysis is pure
Reagent.
(2) myeloma cell prepares: fusion the last week takes out the myeloma cell (SP2/ that a pipe freezes out of liquid nitrogen container
0), be immediately placed in hot water thaw (using it is most be Sp2/0 cell strain, the cell strain growth and fusion efficiencies are good, itself is not
Any heavy chain immunoglobulin or light chain are secreted, the highest growth scale of cell is 9 × 105/ ml, the doubling time is usually 10~
15h；Selection homologous cell strain is considered in practical application relevant to human body, if Shanghai Fu Xiang Biotechnology Co., Ltd is to ATCC
The NCI-H929 human myeloma cell strain that cell bank is introduced).Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min；
It is repeated 1 times.Sediment is moved into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, once passed within 3-4 days
In generation, expands culture, and fusion adjusts cell state in first 24 hours, guarantees that cellular morphology is good before merging, growth is vigorous.It is added
Appropriate basal medium gently beats 1000r/m centrifugation 5-10min after mixing, washes repeatedly cell 2 times into centrifuge tube.
(3) CDl4 cell (macrophage) to be hybridized prepares: the mononuclear macrophage that the present invention sorts is with basal medium
Total cell number is adjusted to 1 × 108~2 × 108For cell fusion.Blue dyeing phase-contrast microscopy, viable count are expected with platform
It should be higher than that 80% is qualified.
(4) cell fusion: CDl4 cell (mononuclear macrophage) and myeloma cell are added with 10: 1-5: 1 ratio
In centrifuge tube, it is mixed evenly, 1000r/m is centrifuged 5min, discards supernatant, and it gently beats tube bottom to cell grainless and precipitates, weight
It is 2 times multiple.Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up
PEG3000 is added drop-wise in fusion pipe in 60s along tube wall while gently rotating centrifugal pipe, later trains the basis of the 25mL of preheating
It supports base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal pipe during addition is then allowed to stand
In 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T culture medium is added.It is inoculated into after appropriate mixing
In 96 well culture plates, 37 DEG C are placed in, is cultivated in 5% CO2 incubator.
(5) the hybridoma mononuclear macrophage strain with phagocytic function is screened: cell grows feelings in 96 well culture plates of observation
Condition, division can be grown by only having hybridoma after 7-10 days, discarded HAT culture medium at this time, replaced complete medium.Cell gram
When grand growth area reaches 1/10 cell hole, culture supernatant is gone, selection has the training of the good hybridoma cell strain of growth conditions
Hole is supported, position, the size of cell strain growth are marked under microscope, cell clone is drawn in the position of mark using sterile pipette tips and arrives
New has in the culture hole of complete medium, and then successively doubling dilution to hole is counted below, and 37 DEG C, 5%CO2 incubator is interior to be cultivated
One week or so, microscopically observation cell growth status, when cell clone is covered with to 1/10 or more hole floor space, take cell or
Culture supernatant detects hybridoma macrophage (M φ) strain function.The specific method is as follows:
1. hybridoma macrophage strain swallows bacterium Function detection: macrophage and staphylococcus or Candida albicans are hanged
Liquid mixing incubates, and smear is fixed, the dyeing of serge blue liquid, in oily phagocytosis situation under the microscope, counts phagocytosis bacterium and does not gulp down
The number of macrophages ratio of bacterium is bitten, to swallow the strong macrophage of bacterium function alternately positive clone strain.
2. hybridoma macrophage strain swallows HIV Function detection: AIDS (AIDS) patient's for taking Disease Control and Prevention Center to save
After blood plasma and hybridoma macrophage strain mixed culture, cell strain is separated, PBS is cleaned 3 times, measures the phagocyte strain through cracking
The function of swallowing HIV, with specific reference to HIV-1p24 antigen detection kit, (enzyme-linked immunization, Shanghai inspire biotechnology limited
Company) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml,
The p24 antigen of 80pg/ml is lower than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity as control, minimum detection limit
450nm measures absorbance (OD) in 0.5pg/ml~80pg/ml, 15min, and blank control calibration object absorbance value is not higher than
0.050,0pg absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbance, is recognized as absorbance > 0.12
To be positive.Specifically operated by kit specification.
3. hybridoma macrophage strain generates macrophage cytokines detection: with human macrophage migration inhibitory factor (MIF)
The operation of ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) by specification, detection range are 0~800pg/ml,
Susceptibility is 1.0pg/ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive stronger, negative
To be colourless or extremely shallow, the depth of the be in color of foundation is indicated with "+", "-" number for reaction.OD value can also be surveyed: in ELISA detector
On, at 450nm (if developing the color with ABTS, 410nm), each hole OD value is surveyed after returning to zero with blank control wells, if more than defined
2.1 times of negative control OD value, it is as positive, specifically operated by kit specification.MIF be collection cell factor, growth factor,
The multi-effect protein molecular of hormone and enzyme characteristic plays central as inherent immunity and the regulatory factor of inflammatory reaction
Effect, it is various infection and active chronic inflammation disease in play panimmunity function.
According to testing result, select the cell clone in the culture hole with stronger macrophage function repeat it is next
Wheel dilution culture, repeats 2-3 wheel, and detection function is taken out after stablizing, and is transferred to culture bottle mass propgation.
(6) preservation and recovery of hybridoma macrophage strain: preceding 12 hour adjustment cell growth state is saved, one bottle of life is taken
Long vigorous, the good cell of form, is made cell suspension after appropriate digestion, 1000r/min is centrifuged 5min, removes supernatant, flick
Tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and dispenses cell cryopreservation tube, and 1mL/ is managed, and -70
Cryopreservation tube, is put into liquid nitrogen container after taking-up and saves backup by DEG C refrigerator overnight.40 DEG C or so of hot water is got out before recovery, it will
Cryopreservation tube carefully takes out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, and is centrifuged after defrosting in 1000r/min
5min opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting washed once with complete culture solution, then
It is centrifuged 5min in 1000r/min, is discarded supernatant, in case making to expand culture.
6, the amplification of hybridoma macrophage strain
That is the industry culture of hybridoma macrophage strain.Above-mentioned cell precipitation is gently resuspended using complete culture solution and is moved back
Enter in culture bottle, sets 37 DEG C, cultivated in 5%CO2 incubator.Amplification cultivation is passed on repeatedly, until required hybridoma cell strain
In quantity, every 10 generation of secondary culture positive hybridoma cell strain, detect the function of hybridoma macrophage strain, see whether to stablize.
Continuation carries out extensive industrialization preparation in several bottles.
7, the preparation (preparation for the treatment of AIDS organelle) of hybridoma macrophage strain organelle
By the hybridoma macrophage strain of (above-mentioned) preparation of the present invention, after being cleaned with physiological saline, then with 1000r/min from
Heart 5min (low speed is centrifuged in short-term) takes cell precipitation to be packed into cylinder made of acrylate etc high-biocompatibility material and describes
In device, sterile saline is then filled it up with, makes the concentration 80%~90% of hybridoma macrophage strain, is sealed, is made immediately
The treating AIDS organelle used.Hybridoma macrophage strain in organelle, which has, to be swallowed and adsorbs HIV and secrete huge
The function of the phagocyte factor, the shape of organelle can be made into infundibulate, and bottom diameter is small, and top diameter is big, and volume is 200~300ml, disengaging
Mouth is equipped with cell screen clothes, and entrance top diameter sieve mesh number is 800 mesh；Exit bottom diameter sieve mesh number is 2.0~5.0 mesh (2.5
~5.0 mesh are equivalent to 0.1~0.2 micron or 100~200 nanometers), specifically can be made into 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh,
The specification of 4.0 mesh, 4.5 mesh and 5.0 mesh, to stop 120 nanometers inhibition of HIV or bigger bacterium；Liquid outlet setting
Mesh number is the cell strainer of 100 mesh (being equivalent to 4 microns), to the cell for stopping to filter out；Liquid entrance and mesh screen it
Between be equipped with buffer area, be conducive to the stability of system circulation.When the blood plasma separated through extracorporeal circulation apparatus flows through organelle, trip
From HIV by corresponding hybridoma macrophage strain swallow adsorb, purified blood plasma from organelle flow out, separated with separator
Haemocyte converge after feed back it is internal.Treating AIDS organelle of the invention can entrust professional businessman to prepare.
8, the preparation of plasma separator
(1) it preparation principle: is prepared according to the molecular size of haemocyte and blood plasma components.Such as visible component in blood of human body
The size of (haemocyte) are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds,
About 12 μm of neutrophil leucocyte, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, small lymphocyte 6-
8 μm, approximate with red blood cell, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, 1~4 micron to 7~8 microns of diameter
It differs, the platelet mean diameter of people is 2-4 microns, 0.5~1.5 micron thick.
(2) poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. material: can be selected, it is desirable that good biocompatibility hardly swashs
Living complement, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.It can be by altogether
The methods of valence, grafting, polymerization improve the structure of material, the microinhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxygen
Change stress influence, the generation to improve sieving adequacy and biocompatibility, reduce complication.
(3) type and spec: for the shape of separator, filter core preparation can be made with materials such as acetate fiber or absorbent cotton
The shapes such as flat structure are prepared into as filter core at column construction, with materials such as poly-vinegar non-woven fabrics；By haemocyte and blood to be separated
The molecular size of slurry composition determines aperture.It is plasma separator according to the present invention property stabilization, good biocompatibility, penetrating
Property high high molecular polymer hollow fibre type filter is made, hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm,
Aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm.The hole only permit blood plasma filtration, but can stop all cells at
Point.
9, the application for the treatment of AIDS organelle
By the application schematic diagram of Fig. 1 in the present invention, plasma separator and organelle are connected, constitutes extracorporeal circulation apparatus, tool
Body method is as follows:
(1) it installs: with sterile working connecting components, including plasma separator, organelle and each circulation line.
(2) it is vented: with sterile saline filling liquid separator, organelle and each circulation line, excluding separator, organelle
And its gas, bubble in circulating line, it goes through, confirmation after gas, bubble without using.
(3) lead to liquid: arterial blood line pipe 1 being connected to the arteries of AIDS patient, in operation the row of going through again
Whether gas is complete, and whether liquid stream is unobstructed, and flow liquid in pipe is avoided to pollute.
(4) anticoagulant: to be injected from heparin pump into liquid stream anti-coagulants (heparin), be for the first time 2500 ∪ or 20~30 ∪/kg.
(5) start: venous line (5) being connected to the vein blood vessel of AIDS patient, then open blood pump, blood flow is
100~150ml/min, such as Fig. 1 are separated when arterial blood enters plasma separator (4) through arterial blood line pipe (1)
Blood plasma reaches organelle (8) through circulation line (7) under the action of blood plasma pump 6, wait be full of blood plasma, about 10 minutes, begins releasing
Blood plasma is flowed out through circulation line (10), synchronous that blood plasma is perfused to organelle (9), and the blood plasma in organelle (8) has nearly flowed
When, perfusion blood plasma is started again at, organelle (9) begins releasing blood plasma at this time, and two organelles (8) in parallel, organelle (9) are handed over
For progress.Such as Fig. 2, when blood to be separated enters inner cavity (2) of plasma separator (1), the effect through valve (8) can lead to
The small molecule blood plasma components (5) for crossing micropore (3) enter the exocoel (6) of separator, then flow out through plasma outlet port (7), and cannot
It is flowed out by the haemocyte (4) of micropore (3) through valve (8).Such as Fig. 3, when the blood plasma containing HIV (3) enters organelle (1),
HIV (3) therein be fixed on the hybridoma macrophage strain (2) in organelle phagocytosis absorption, formed compound (4) and no longer
It moves down, and the cell factor (5) that hybridoma macrophage strain generates is blended in the blood plasma after removing HIV and flows out cell
After device, fed back through venous line shown in FIG. 1 (5).So until the plasma circulation amount (usually 9L) being previously set, treatment is
It ends.If mating computer program control, entire therapeutic process is controlled by computer, and can detect working condition at any time,
, automation more convenient using meeting and safety.
10, the additional component for the treatment of AIDS organelle
The treating AIDS organelle that Fig. 1 is provided is basic composition part of the invention, can also be reequiped on this basis
With lower component, including sound pulse pressure and air monitering, temperature control system, liquid mixing system, off gas system, monitored conductivity system
The parts such as system, ultrafiltration monitoring and leakage blood monitoring.
(1) sound pulse pressure monitors: in addition the main stopping state to dynamic monitoring organelle micropore of arterial blood pressure monitoring is used
To monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When blood flow deficiency, angiosthenia will be reduced；When having blood coagulation, thrombus
When formation, especially separator blockage of the micro orifice, angiosthenia will be increased；Vein pressure monitoring is used to monitor the pressure of pipeline blood reflux
Power, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow deficiency and venous return syringe needle fall off, vein pressure will
Decline, if bloody path return pipe distortion blocking or reflux syringe needle block, vein pressure will be increased.
(2) air monitering (Air Detector): the air bubble for monitoring blood pathway generally uses ultrasonic listening
Principle, in order to avoid patient occur air embolism and be arranged.When having monitored air bubble, detection system can drive it is dynamic,
Vein bloody path folder carrys out blocking blood flow, prevents dangerous generation.
In short, on the basis of basic component system of the present invention, it is expected to further research and develop automation therapeutic equipments, develops and be
The hommization of operation, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily
Judge the micro computers processing system such as alarm reason and ring off signal.
11, experimental verification
The effect of to understand effect of the invention, devising Simple cell device test method: take 2.5 × 300mm of sterilizing
Westergren's blood sedimentation tube 5, draw respectively be centrifuged (1000r/min, 5min) precipitating the strain of hybridoma macrophage to 200mm carve
Degree then draws the heat preservation after 100 DEG C dissolve and reaches about 10mm long scale in 56 DEG C of 0.9% spare agarose C1-4B, set
After blood sedimentation stand is cooling, agarose becomes semisolid, and blood sedimentation tube inner cell can be prevented to flow out but not prevent water and the change of small molecule
The substance for studying part etc passes through.5 blood for AIDS (AIDS) patient that Disease Control and Prevention Center of the Zhejiang Province Ling Qu equal samples library saves
Slurry, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and injects blood sedimentation tube (Simple cell device) upper end blank pipe, blood to be flowed through in batches
The hybridoma macrophage strain layer of immersed tube lower layer simultaneously after outflow, collects efflux, blood plasma after referred to as AIDS filter out of blood sedimentation tube.It takes
Blood plasma and blood plasma after filter before AIDS is filtered, with human macrophage migration inhibitory factor (MIF) ELISA detection kit (hundred stamen of Shanghai
Biotechnology Co., Ltd) pairing detection, by specification operation, detection range is 0~800pg/ml, susceptibility 1.0pg/
Ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive stronger, and negative reaction is colourless or pole
Shallowly, it according to the depth of be in color, is indicated with "+", "-" number.OD value can also be surveyed: on ELISA detector, in 450nm (if with
ABTS develops the color, then 410nm) at, each hole OD value is surveyed after returning to zero with blank control wells, if more than defined negative control OD value
It is 2.1 times, as positive.As a result such as table 1, MIF testing result is negative (or because content is sensitive lower than detecting in blood plasma before filtering
Degree, blood plasma long-term preservation cause degradation etc.), and testing result is the positive in blood plasma after filtering, and illustrates macrophage hybridoma
Strain produces MIF cell factor in this process.MIF is to integrate cell factor, growth factor, the multiple-effect of hormone and enzyme characteristic
Can protein molecular, the effect of central is played as inherent immunity and the regulatory factor of inflammatory reaction, in various infection and anxious slow
Property diseases associated with inflammation in play panimmunity function.MIF pairing detection is same before and after making AIDS blood plasma filtration Simple cell device
When, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 antigen detection kit (enzyme-linked immunization, Shanghai
Inspire Biotechnology Co., Ltd) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml,
As control, minimum detection limit is lower than 5pg/ml for the p24 antigen of 20pg/ml, 40pg/ml, 80pg/ml, and measurement range 0~
450nm measures absorbance (OD) in 400pg/ml, the range of linearity 0.5pg/ml~80pg/ml, 15min, blank control calibration object
Absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbance not higher than 0.050,0pg absorbance value, works as extinction
Be considered as when spending > 0.12 it is positive, after testing result (table 2) illustrates AIDS blood plasma filtration Simple cell device, part HIV by
Hybridoma macrophage strain phagocytosis absorption, the blood plasma HIV after filtration are significantly reduced, and after the 1st filtration, HIV clearance rate is
20.55%, after the 2nd filtration, HIV clearance rate is 42.83%, p < 0.01, has significant effect, illustrates with filtration
The increase of number, HIV can be removed constantly, to reach treatment AIDS purpose.
MIF pairing testing result is (quantitative: pg/ml) before and after 1 AIDS blood plasma of table filters Simple cell device
2 AIDS blood plasma of table filters p24 testing result (p24:pg/ml) before and after Simple cell device
In short, from Tables 1 and 2 the result shows that, the blood plasma containing HIV is filtered through treating AIDS organelle of the invention
Afterwards, HIV therein can be adsorbed by the hybridoma macrophage strain in organelle and be removed, significant effect；At the same time, hybridoma is huge
Phagocyte strain is produced with anti-infective and immunization cell factor.The experiment results show that treating AIDS of the invention
Organelle has significant effect.
Claims (13)
1. a kind for the treatment of AIDS organelle for field of biomedicine, which is characterized in that including high-biocompatibility material
And hybridoma cell strain, the hybridoma cell strain is wrapped up with high-biocompatibility material, is constructed to prevent cell and its fragment
And HIV filters out and can swallow for hybridoma cell strain HIV and generate cell factor and provides the organelle in place；With primary cell
Cd4 cell therein is sorted, after cultivation, CD14 cell is further sorted, is followed by prepared into the CD14 cell sorted both
Retain archaeocyte characteristic and the hybridoma cell strain of energy infinite multiplication, the hybridoma cell strain can swallow HIV and generate thin
Intracellular cytokine.
2. treating AIDS organelle according to claim 1, which is characterized in that the primary cell includes single core blood
Liquid cell, single core histocyte, amniocyte and/or villus cell.
3. treating AIDS organelle according to claim 1, which is characterized in that the method for sorting CD14 cell includes exempting from
Epidemic disease paramagnetic particle method, adherent method, flow cytometry.
4. treating AIDS organelle according to claim 2, which is characterized in that the method for sorting CD14 cell includes exempting from
Epidemic disease paramagnetic particle method, adherent method, flow cytometry.
5. treating AIDS organelle according to claim 1, which is characterized in that the hybridoma cell strain is by myeloma
Cell SP2/0 or human myeloma cell strain are prepared.
6. treating AIDS organelle according to claim 2, which is characterized in that the hybridoma cell strain is by myeloma
Cell SP2/0 or human myeloma cell strain are prepared.
7. treating AIDS organelle according to claim 3, which is characterized in that the hybridoma cell strain is by myeloma
Cell SP2/0 or human myeloma cell strain are prepared.
8. treating AIDS organelle according to claim 4, which is characterized in that the hybridoma cell strain is by myeloma
Cell SP2/0 or human myeloma cell strain are prepared.
9. -8 any treating AIDS organelle according to claim 1, which is characterized in that the volume of organelle is 200
~300ml, is equipped with top diameter cell screen clothes at liquid-inlet, and top diameter cell screen clothes mesh number is 800 mesh, liquid-inlet with push up diameter cell
The buffer area that promotion system stablizes circulation is equipped between sieve, exit is equipped with bottom diameter cell screen clothes and cell strainer, and bottom diameter is thin
Born of the same parents' sieve mesh number is 2.0~5.0 mesh, and cell strainer mesh number is 100 mesh, is also equipped between bottom diameter cell screen clothes and liquid outlet slow
Area is rushed, the stability of system circulation is conducive to.
10. treating AIDS organelle according to claim 9, which is characterized in that organelle exit bottom diameter sieve mesh
Numeral system at 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh 7 kinds of different sizes.
11. -8,10 any treating AIDS organelle according to claim 1, which is characterized in that the organelle and blood
It starches separator to be connected, the plasma separator is hollow fibre type, and diameter is 270~370 μm, film thickness is 50 μm, aperture is
0.2~0.6 μm, length be 13.5~26 μm, can stop every other blood constituent, only allowance blood plasma filter.
12. treating AIDS organelle according to claim 9, which is characterized in that the organelle and plasma separator
It is connected, the plasma separator is hollow fibre type, and diameter is 270~370 μm, film thickness is 50 μm, aperture is 0.2~0.6 μ
M, length is 13.5~26 μm, can stop every other blood constituent, only permits blood plasma filtration.
13. any treating AIDS organelle of claim 1-12 is preparing the application in extracorporeal blood circulating device,
It is characterized in that, the extracorporeal blood circulating device includes arterial blood line pipe (1), one end is through heparin pump (2) and blood pump (3)
It is connected with plasma separator (4), plasma separator (4) first cell in parallel with two through blood plasma pump (6) and circulation line (7)
Device (8), the second organelle (9) are connected, and are then successively connected with circulation line (10), venous line (5).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610540997.9A CN106178162B (en) | 2016-07-01 | 2016-07-01 | Treating AIDS organelle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610540997.9A CN106178162B (en) | 2016-07-01 | 2016-07-01 | Treating AIDS organelle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106178162A CN106178162A (en) | 2016-12-07 |
| CN106178162B true CN106178162B (en) | 2018-12-04 |
Family
ID=57473976
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610540997.9A Active CN106178162B (en) | 2016-07-01 | 2016-07-01 | Treating AIDS organelle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106178162B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109172907A (en) * | 2018-07-01 | 2019-01-11 | 翁炳焕 | A kind of AIDS immunoadsorption therapy instrument |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006520756A (en) * | 2003-01-29 | 2006-09-14 | パナコス ファーマシューティカルズ インコーポレーティッド | Inhibition of HIV-1 replication by disruption of viral capsid spacer peptide 1 protein processing |
| US20090081176A1 (en) * | 2007-09-07 | 2009-03-26 | Rudolph Maravich | Cure for the human immunodeficiency virus |
| CN103170023A (en) * | 2011-12-22 | 2013-06-26 | 张涛 | AIDS therapeutic equipment and manufacturing method |
| CN102631891A (en) * | 2012-04-23 | 2012-08-15 | 武汉大学 | Human immunodeficiency virus affinity adsorption column, and preparation method and uses thereof |
| CN103691016A (en) * | 2014-01-15 | 2014-04-02 | 倪自谦 | Aids virus specific plasma adsorption column and application method thereof |
| CN104801278A (en) * | 2015-03-29 | 2015-07-29 | 武汉瑞法医疗器械有限公司 | Preparation method of AIDS (acquired immune deficiency syndrome) virus affinity adsorbent |
-
2016
- 2016-07-01 CN CN201610540997.9A patent/CN106178162B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106178162A (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106267422B (en) | Rh blood group incompatibility haemolysis disease therapeutic apparatus | |
| CN106267410B (en) | HIV infection cell separator | |
| CN106267413B (en) | AIDS plasma purification device | |
| CN106110425B (en) | AIDS plasma purification therapeutic equipment | |
| CN106178162B (en) | Treating AIDS organelle | |
| CN106178163B (en) | AIDS biological cell immunization therapy instrument | |
| CN106267425A (en) | AIDS immunoadsorption therapy instrument | |
| CN106075626B (en) | A kind of AIDS blood purifying therapeutical instrument | |
| CN106267416B (en) | AIDS therapeutic equipment | |
| CN106039448B (en) | AIDS cell adsorbing therapy instrument | |
| CN106166313B (en) | A kind of AIDS pregnant woman blood clarifier | |
| CN106267419B (en) | HIV immunologic purging device | |
| CN106267411B (en) | AIDS toxin expelling therapeutic equipment | |
| CN106267420B (en) | HIV phagocyte device | |
| CN106267406B (en) | AIDS blood purification | |
| CN106344986B (en) | Female tire blood group incompatibility treats absorber | |
| CN106267415B (en) | AIDS purification treatment instrument | |
| CN106267412B (en) | AIDS immunologic purging instrument | |
| CN106267404B (en) | AIDS biological cell therapeutical instrument | |
| CN106110426B (en) | AIDS immunization therapy instrument | |
| CN106267409B (en) | AIDS biological therapy reactor | |
| CN106110422B (en) | AIDS immunization therapy absorber | |
| CN106267417B (en) | AIDS therapeutic response device | |
| CN106267414B (en) | AIDS immunologic purging device | |
| CN106267424B (en) | AIDS immune cell therapy instrument |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190322 Address after: Room 405 and 501, Building 672, Huazhong South Road, Xiacheng District, Hangzhou City, Zhejiang 310000 Patentee after: Hangzhou co Genesis Laboratory Laboratory Limited Address before: 311100 Zhejiang Province Hangzhou Yuhang District Cangqian Street Luting Road No. 1, Building 3, Room 113, Zhejiang Tongchuang Medical Laboratory Center Co., Ltd. Patentee before: Weng Binghuan |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201102 Address after: No.1, Chengguan Shuige Road, Xianju County, Taizhou City, Zhejiang Province, 317300 Patentee after: Weng Binghuan Patentee after: HANGZHOU TONGCHUANG MEDICAL EXAMINATION LABORATORY Co.,Ltd. Address before: 310000 room 672, No. 405 and 501, No. 672, No. 3, Huazhong Nan Lu, Xiacheng City, Zhejiang Patentee before: HANGZHOU TONGCHUANG MEDICAL EXAMINATION LABORATORY Co.,Ltd. |