CN101143207B - Application of natural type, breaking type or No. 5 domain delation variant type beta 2 glycoprotein I in preparing medicine for inhibiting blood vessel newborn - Google Patents
Application of natural type, breaking type or No. 5 domain delation variant type beta 2 glycoprotein I in preparing medicine for inhibiting blood vessel newborn Download PDFInfo
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Abstract
The invention discloses the application of a beta-2-glucoprotein-I of the natural type, the cracking type or the V structure domain deletion mutant type in preparing a novel drug of restraining the angiogenesis. The test testifies that the natural type beta-2-glucoprotein-I (n beta2GPI), the cracking type beta-2-glucoprotein-I (c beta2GPI) and the V structure domain deletion mutant type beta-2-glucoprotein-I (DI-IV) can restrain the angiogenesis of the endothelial cell. The functional mechanism of the invention is that the invention influences the expression and photophosphorylation of an accepter of an endothelial cell growth factor to retain the signal conducting path of the accepter and retain the lower fifty genetic expressions and further retain the multiplication, the migration and the angiogenesis of the endothelial cell. The invention can provide novel preventing and remedying measures for clinical treatment of the diseases of the angiogenesis, such as the multiplication type retina lesion of the diabetes etc.
Description
Technical field
The present invention relates to the purposes of beta 2 glycoprotein I, particularly relate to the purposes of beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
Background technology
Angiogenesis is called blood vessel generation, angiogenesis or vascularization again, is to sprout or branched mode grows the process of neovascularity from the body blood vessel.Normal vascularization plays an important role in fetal development, wound repair and menstrual cycle.Vascularization mainly comprises four-stage: (1) vascular endothelial cell dedifferentes, and the vascular permeability increase also seepage occurs; (2) endotheliocyte discharges protein resolvase, degradation of cell epimatrix, broken cyclic group counterdie; (3) migration of endotheliocyte and propagation; (4) endotheliocyte forms tubular structure and interconnects etc.
[5]Blood vessel is subjected to the regulation and control of multiple positivity and negativity regulatory factor.Have now found that the various kinds of cell factor is relevant with vascularization, wherein promote the angiopoietic factor have VEGF (vascular endothelial growth factor, VEGF), basic fibroblast growth factor (bFGF) etc.; The factor of angiogenesis inhibiting has thromboxane (thrombospondin), angiostatin etc.Under the physiological status, angiogenesis factor and inhibitive factor are in poised state, and endotheliocyte is in static relatively.Under some pathological conditions, the balance of the two is destroyed.Wherein the overexpression of angiogenesis factor can cause unusual vascularization, often with diseases such as tumor, diabetic renal papillary necrosis, atherosclerosis, rheumatoid arthritis, psoriasiss substantial connection is arranged.
It is one of main ophthalmic of diabetics blinding that PDR becomes (PDR), is the focus of clinical concern to its control.Studies confirm that retinal vessel new life is the arch-criminal of PDR morbidity unusually, and VEGF (VEGF) is the important factor of angiogenesis promoting.At present often use treatment PDR such as laser photocoagulation and vitrectomy, but severe complication such as postoperative detachment of retina may take place.Found that in addition the chimeric protein of vegf receptor, anti-VEGF monoclonal antibody etc. can suppress intraocular neovascularization and form, but had problems such as complicated operation, cost height, inhibition degree be irregular.Therefore seek a kind of safe, effective, economic medicine of preventing VEGF and expression of receptor and function, be significant for diabetic renal papillary necrosis, tumor, atherosclerotic control.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, the application of a kind of natural type beta 2 glycoprotein I in the preparation anti-angiogenic drugs is provided.
Second purpose of the present invention provides the application of a kind of breaking type beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
The 3rd purpose of the present invention provides the application of a kind of V domain deletion mutation type beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
Technical scheme of the present invention is summarized as follows:
The application of a kind of natural type beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
The application of a kind of breaking type beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
The application of a kind of V domain deletion mutation type beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
Description of drawings
Fig. 1 is natural type beta 2 glycoprotein I (n β
2GPI), breaking type beta 2 glycoprotein I (c β
2GPI), V domain deletion mutation type beta 2 glycoprotein I (DI-IV) suppresses the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC) propagation;
Fig. 2 is natural type beta 2 glycoprotein I (n β
2GPI), breaking type beta 2 glycoprotein I (c β
2GPI), V domain deletion mutation type beta 2 glycoprotein I (DI-IV) suppresses the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) propagation;
Fig. 3 is natural type beta 2 glycoprotein I (n β
2GPI) suppress the migration of VEGF (VEGF) and the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC);
Fig. 4 is natural type beta 2 glycoprotein I (n β
2GPI), breaking type beta 2 glycoprotein I (c β
2GPI), V domain deletion mutation type beta 2 glycoprotein I (DI-IV) suppresses VEGF (VEGF) and the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) migration;
Fig. 5-1 is natural type beta 2 glycoprotein I (n β
2GPI) (1 μ M) suppresses the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC) vascularization;
Fig. 5-2 is natural type beta 2 glycoprotein I (n β
2GPI) (1 μ M) suppresses the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) vascularization;
Fig. 5-3 is breaking type beta 2 glycoprotein I (c β
2GPI) (1 μ M) suppresses the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC) vascularization
Fig. 5-4 is breaking type beta 2 glycoprotein I (c β
2GPI) (1 μ M) suppresses the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) vascularization
Fig. 5-5 is that V domain deletion mutation type beta 2 glycoprotein I (DI-IV) (1 μ M) suppresses the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC) vascularization;
Fig. 5-6 is that V domain deletion mutation type beta 2 glycoprotein I (DI-IV) (1 μ M) suppresses the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) vascularization;
Fig. 5-7 is that I domain deletion mutation type beta 2 glycoprotein I (DII-V) (1 μ M) does not influence the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC) vascularization;
Fig. 5-8 is that I domain deletion mutation type beta 2 glycoprotein I (DII-V) (1 μ M) does not influence the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) vascularization;
Fig. 5-9 does not influence the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC) vascularization for human serum albumin (HSA) (1 μ M);
Fig. 5-10 does not influence the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) vascularization for human serum albumin (HSA) (1 μ M);
Fig. 5-11 is for only there being Human umbilical vein endothelial cells under the culture fluid situation (HUVEC) vascularization (contrast);
Fig. 6 is natural type beta 2 glycoprotein I (n β
2GPI), breaking type beta 2 glycoprotein I (c β
2GPI), V domain deletion mutation type beta 2 glycoprotein I (DI-IV) suppresses the mean vascular length of the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC);
Fig. 7 is natural type beta 2 glycoprotein I (n β
2GPI), breaking type beta 2 glycoprotein I (c β
2GPI), V domain deletion mutation type beta 2 glycoprotein I (DI-IV) suppresses the mean vascular length of the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC);
Fig. 8 is natural type beta 2 glycoprotein I (n β
2GPI) VEGF (VEGF) receptor mRNA that suppresses VEGF (VEGF) and the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) is expressed;
Fig. 9 is natural type beta 2 glycoprotein I (n β
2GPI) phosphorylation that suppresses the interior Akt of the inductive Human umbilical vein endothelial cells of VEGF (VEGF) (HUVEC), ERK (1/2), GSK3B (Ser9) activates;
Figure 10 is natural type beta 2 glycoprotein I (n β
2GPI) phosphorylation that suppresses the interior Akt of the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC), ERK (1/2), GSK3B (Ser9) activates;
Figure 11 is that relative ribbon density analysis confirms natural type beta 2 glycoprotein I (n β
2GPI) phosphorylation that suppresses VEGF (VEGF) and the interior Akt of the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) activates;
Figure 12 is that relative ribbon density analysis confirms natural type beta 2 glycoprotein I (n β
2GPI) phosphorylation that suppresses VEGF (VEGF) and the interior GSK3B of the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) activates;
Figure 13 is that relative ribbon density analysis confirms natural type beta 2 glycoprotein I (n β
2GPI) phosphorylation that suppresses VEGF (VEGF) and the interior ERK of the inductive Human umbilical vein endothelial cells of basic fibroblast growth factor (bFGF) (HUVEC) (1/2) activates.
The specific embodiment
We are by Matri glue and VEGF/bFGF (VEGF is a VEGF, and bFGF is a basic fibroblast growth factor) research natural type β
2GPI, breaking type β
2GPI or domain deletion mutation type β
2Whether GPI is in external angiogenesis generation effect to huve cell.
Our result shows natural type beta 2 glycoprotein I (n β
2GPI), breaking type beta 2 glycoprotein I (c β
2GPI) and V domain deletion mutation type beta 2 glycoprotein I (DI-IV) can by domain I at vitro inhibition VEGF and the inductive angiogenesis of bFGF, this is to finish by the expression of downward modulation endotheliocyte KDR/Flk-1 and then the phosphorylation of blocking-up MAPK/ERK and PI3K/Akt/GSK3 β path middle and lower reaches effector molecule, and I domain deletion mutation type beta 2 glycoprotein I (DII-V) does not have this effect.
Materials and methods
Reagent
Is the aminoacid sequence of natural beta 2 glycoprotein I, V domain deletion mutation type beta 2 glycoprotein I (DI-IV) and I domain deletion mutation type beta 2 glycoprotein I (DII-V) seen http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=protein﹠amp; Id=28814, serial number are CAA40977.
Natural β 2GPI (the n β in blood plasma source
2GPI) available from Haematologic Technologies Inc (EssexJunction, VT).
The preparation method of several beta 2 glycoprotein Is:
The expression and purification of (one) V domain deletion mutation type beta 2 glycoprotein I (DI-IV) and I domain deletion mutation type beta 2 glycoprotein I (DII-V):
1. make up the baculovirus vector that contains with histidine-tagged target gene fragment
(1) pcr amplification genes of interest:
V domain deletion mutation type beta 2 glycoprotein I (DI-IV):
Forward primer: 5 '-GTAATAAAAAACCTATAAAT-3 '
Downstream primer: 5 '-ACAGAATTCTTAACAACTTGGCATGGCAGA-3 '
I domain deletion mutation type beta 2 glycoprotein I (DII-V)
Forward primer: 5 '-ACTCTGAATTCTACACCCAGAGTATGT-3 '
Downstream primer: 5 '-GTAAAACGACGGCCAGT-3 ' (M13 primer)
Reaction condition: 95 ℃ of degeneration 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 3min, 25 circulations.The 100ul reaction system (contains the 10mMTris/HCl buffer, pH8.3,50mM KCl, 1.5mMMgCl
2, 0.01% gelatin, 200 μ MdNTP, 1 μ M upstream downstream primer, 3Utaq enzyme).
(2) genes of interest is connected with carrier
Prepare V domain deletion mutation type beta 2 glycoprotein I (DI-IV) and digest genes of interest respectively with BamHI/EcoRI, with plasmid pVL1393, use ligase to make up the recombinant baculovirus (abbreviating pVLLD15 and pVLLD14 respectively as) that comprises target gene fragment.
Prepare I domain deletion mutation type beta 2 glycoprotein I (DII-V) and digest genes of interest with BamHI/EcoRI, use EcoRI/BglII digested plasmid pVLL1 simultaneously, use ligase to make up the recombinant baculovirus (abbreviating pVLLD25 as) that comprises target gene fragment.
(3) High Five
TMExpress target protein in the insect cell line:
27 ℃ of 100RPM of Express Five SFM culture fluid cultivate sf9 (fall army worm) cell, reach 1-2x10
6Behind cell/ml, every 1000ml adds the 30ml viral vector, extracts various types of beta 2-glycoprotein I after 50 hours.
2. use the Ni-NTA resin to extract purification of target albumen
Principle: the poly histidine can combine with multiple transition metal and transition metal chelate, and therefore the albumen of the His-6 of band exposure can be incorporated into solidified Ni
2+Resin, behind suitable other protein of buffer flushing removal, but the soluble competitive chelating agen eluting of reuse can reclaim target protein.Efficient, high power capacity, powerful concentrating and characteristics are fast arranged.
A) preparation Ni
2+Affinity column.
B) extract the supernatant that contains target protein.
C) the histidine-tagged target protein of purification band.
Use Nickel bead (Qiagen), binding buffer liquid (20mmol/L sodium phosphate and 500mmol/LnaCl, pH7.8), imidazoles elution buffer (10-150mmol/L), dcq buffer liquid (50mMNaxPO4,0.5mMNaCl, pH7.5-8.0) etc.
3. or with antibody affinity chromatography extract purifying protein: (summary)
4. transform sf9 insect cell (as cell cycle regulation, improve translation post-treatment ability, change the cell metabolism characteristic, realize genome focus combination etc.), improve recombiant protein output and purity (target purity〉95%).
(2) extraction and purification of natural type beta 2-glycoprotein I (n β 2GPI) is identified:
1.17.5% cross the chloric acid precipitation.
2. buffer-exchanged is used the G-25 exchange column.0.05MNaCl0.035M acetate buffer (pH4.8)
3. cation-exchange chromatography.0.05MNaCl0.035M sodium acetate buffer (pH4.8), 0.05MNaCl0.035M sodium acetate buffer (pH5.2) 1MNaCl0.035M sodium acetate buffer (pH5.2)
4. heparin affinity chromatography.0.03MNaCl0.02Mtris-HCl buffer (pH8.0), 0.15MNaCl0.02Mtris-HCl buffer (pH8.0), 0.5MNaCl0.02Mtris-HCl buffer (pH8.0)
5. gel filtration.0.5MNaCl0.02Mtris-HCl buffer (pH8.0)
6. measure SDS-PAGE under protein concentration, the reducing condition, directly ELISA experiment.
7. the growing amount of beta 2-glycoprotein I in the calculating human plasma.
8. amino acid sequencing is identified.
(3) breaking type beta 2 glycoprotein I (c β
2GPI) preparation
Below all enzymatic reactions all contain 20mmol/LNaCl and 0.3mmol/LCaCl at 37 ℃ of 0.1mmol/LTris-HCl (pH7.5)
2Under carry out.Beta 2-glycoprotein I and thrombin react with the molar concentration rate of 56:1, add the guanidine hydrochloride cessation reaction of the 8mmol/L of 2 times of volumes.Buffer with 10mmol/Ltris-HCl (pH8.0) dilutes 10 times, and application of sample is gone into the HiTrap heparin column, to contain the NaCl eluted protein of 0.1mmol/L and 1mmol/L, calculates the growing amount of breaking type beta 2-glycoprotein I respectively.Albumen is identified in the-terminal amino acid order-checking.
Culture fluid of endothelial cell EGM Bulletkit (include endothelial basal medium, 0.5ml rhEGF, the 0.5ml hydrocortisone, 0.5ml GA-1000 (gentamycin, amphotericin B), 2ml Medulla Bovis seu Bubali extract (BBE), 10ml hyclone (FBS),
The endotoxin detection kit available from Clonetics Inc. (Cambrex, MD).M199 culture medium, Hank ' s liquid, phosphate buffer (PBS), hyclone (FBS), penicillin, streptomycin, endothelial cell growth factor (ECGF) (ECGP), NuPAGE
TMThe 4-12% polyacrylamide gel,
Green qRT-PCR test kit available from Invitrogen Corporation (Carlsbad, CA).Human serum albumin (HAS), collagenase, gelatin, EDTA, sodium orthovanadate, protease inhibitor,
The haematoxylin buffer available from Chemical Co. (St.Louis, MO).Recombined human VEGF (rhVEGF) (165 aminoacid), recombined human alkalize cell growth factor (rhbFGF) (157 aminoacid) are available from R﹠amp; D systems (Minneapolis, MN).Anti-Erk1/2, Akt, GSK3B, anti-β-actin, anti-mice IgG horseradish peroxidase, anti-rabbit igg horseradish peroxidase antibody are by Cell Signaling Technology (Beverly, MA) monoclonal mouse anti human CD31 antibody is available from DAKO Australia Pty.Ltd. (Botany, NSW, Australia).The Matri glue (no VEGF and FGF2) that lacks somatomedin available from Becton Dickinson Biosciences (Bedford, MA).CellTiter
Aqueous analysis of cell proliferation test kit available from Promega (Madison, WI).
RNA extract test kit available from QIAGEN Pty.Ltd. (Clifton Hill, Victoria, Australia).Rotor-Gene
TM3000 real-time quantitative PCR analysers from Corbett life science (Sydney, NSW, Australia).BCA analysis of protein test kit from Pierce (Rockford, IL).Nitrocellulose filter (0.2 μ m), ECL western trace detectable available from Amersham Biosciences UK Ltd. (Little Chalfont, Bucking hamshire, UK).The separation and the cultivation of Human umbilical vein endothelial cells (HUVECs)
HUVECs obtains from fresh umbilical cord according to the method for having set up.In brief, conduit is inserted in the vein of people's umbilical cord, to remove inner blood, hatched 15 minutes for 37 ℃ with 1mg/ml II Collagen Type VI enzyme then with the flushing of Hank ' the s buffer of pre-cooling.Endotheliocyte is inoculated in 75cm behind the resuspension
2Culture bottle in, containing 5%CO
237 ℃ of incubators in hatch.Behind cell fusion, separate with the digestion of 5% pancreas enzyme-EDTA solution.According to endotheliocyte typical cobblestone sample form and the dyeing of anti-CD31 antibody mediated immunity group it is identified.
Cell is cultivated with the M199 culture medium that contains 20% hyclone, 1% penicillin and streptomycin in stimulation test, then use the M199 culture medium culturing 12 hours of serum-free, and then in serum-free medium, apply intervention factor: the β of natural, fracture, domain deletion form sudden change
2GPI (DI-IV and DII-V), HSA, VEGF and bFGF.All reagent and culture medium all with
The Limulus test kit detects endotoxin.
Anti-CD31 antibody carries out immunohistochemical staining to HUVECs
With 2-3x10
4HUVECs is added in air-dry on the slide, acetone fixed, then with TBS flushing 3 times.Dripping the 100ul3% hydrogen peroxide at room temperature hatched 10 minutes, TBS flushing 3 times, dripping anti-CD31 antibody of 100ul (1:100 dilution) or isotype mice IgG (contrast) hatched 1 hour again, the anti-mice IgG (1:100 dilution) that adds the 100ulHRP labelling after the TBS flushing was again hatched 40 minutes, and TBS cleans 3 times.DAB colour developing, haematoxylin are redyed, the TBS flushing is observed the CD31 positive cell with the microscope imaging system in the back for several times.
External endotheliocyte wound healing experiment
Using external wound healing tests and observes β
2GPI is for the influence of rhVEGF and the inductive HUVECs migration of rhbFGF.48 orifice plates with 0.1% gelatin bag quilt are cultivated HUVECs, behind the cell fusion, with 100-200 μ l micropipette tip cell monolayer is carried out cut and form an exposed area, clean the back with the culture medium of serum-free and cultivate with the culture medium that contains 2% hyclone (this concentration can be kept the migration that cells survival does not still cause cell).Add rhVEGF (50ng/ml) or rhbFGF (20ng/ml), in contrast in the culture medium respectively, and add n β with no somatomedin
2β GPI (100nMand1 μ M), fracture or sudden change
2GPI (DI-IV and DII-V) (100nM) or HSA (albumin human) (100nM and1 μ M), hatch 18 hours after, fix, HE dyeing, carry out microphotograph with twice back of PBS flushing, 10% formalin with low power lens (* 50).Following method is used in the quantitative analysis of cell migration: counting move to the cell number of exposed area from the cut edge and divided by the area of exposed area with cells/mm
2Expression, count in 3 visuals field of every porocyte picked at random.Every group of cell repeats to cultivate 3 holes respectively.
The proliferation experiment of HUVEC
Cell is with 5, the density in 000/hole is inoculated in 96 orifice plates, cultivates after 24 hours with hungry 12 hours of the culture medium of serum-free, cultivates 72 hours with the M199 culture medium that contains 2% hyclone, add rhVEGF (10ng/ml) or rhbFGF (10ng/ml) in the culture medium, add n β simultaneously
2GPI or HSA (10nM, 100nM and1unM).20ulMTS reagent be added drop-wise in each hole after the 100ul culture medium is mixed, hatched 2 hours for 37 ℃.Microplate reader is measured the absorbance of 490nm, and the absorbance of 490nm is directly proportional with the quantity of survivaling cell, and the range of linearity of experiment is 1000 and 8000 cells/well.
External HUVEC blood vessel on Matri glue is tested
Before the cell bed board, 96 orifice plates earlier wrap quilt with the Matri glue that 70ul does not contain somatomedin, and 37 ℃ were cured in 30 minutes.HUVEC is with 0.5x10
4Density be inoculated in Matri glue surface and cultivate in the culture medium of serum-free, do not add or add 10ng/ml rhVEGF or 10ng/ml rhbFGF in the culture medium, and add n β
2GPI, c β
2The rh β of GPI, sudden change
2GPI (DI-IV and DII-V) (concentration is 10nM, 100nM and 1uM).The situation that monitoring blood capillary spline structure forms after 6 hours is taken pictures with low power lens (* 50) and is stored as the TIFF picture with microscopy imaging system.When each the analysis, 3 porocytes of same experiment condition are taken pictures in 5 visuals field of picked at random respectively.What the graphical analysis that the pipe spline structure forms was adopted is the software of downloading from the Internet
Http:// www.scioncorp.com/, image is entered as the Scion picture and converts the form of binary to.The tubule calculation methods of length is to draw a line on each tubule next door then with the length of the form slotted line of pixel.The data of all measurements convert the unit of um to after microscope/camera is measured, be the scale of unit because installed in camera lens with um.
Real-time quantitative PCR measures KDR/Flk-1 and Flt-1mRNA expresses
HUVECs after the overnight incubation, applied the processing factor 60 minutes in the M199 of serum-free culture medium: HAS or n is β
2GPI (2 μ M).Adding 37 ℃ of rhVEGF (40ng/ml) then in the cell hatched 30 minutes.What the extraction of cell total rna was adopted is
RNA blood mini Kits uses RNase-Free Dnase to remove the interference of genomic DNA, and the RNA concentration of being extracted is calculated with the numerical value of OD260nm; The bar that the integrity of RNA is observed ribosome 18s and 28s subunit with EB dyeing with agarose gel electrophoresis brings evaluation.The ratio of OD260/OD280 is at 1.8-2.0.The RNA of 1ug is reversed record and is cDNA, and what primer design adopted is Primer3.0 software, and primer sees that no dimer formation does not have the non-specific amplification product.The primer sequence of KDR/Flk-1 (AF063658) is as follows: upstream 5 '-ACCCCTTGAGTCCAATCACAC-3 ', downstream 5 '-CATGGCTCTGCTTCTTCTCCTTTG-3 '; Flt-1 (NM_002019) upstream: 5 '-TCTCACACATCGACAAACCAATACA-3 ', downstream 5 '-GGTAGCAGTACAATTGAGGACAAGA-3 '.; House-keeping gene β-actin (BC013835) upstream 5 '-CTGGAACGGTGAAGGTGACA-3 ', downstream 5 '-AAGGGACTTCCTGTAACAATGCA-3 '.The real-time quantitative PCR system comprises: 12.5ul
Green qPCR superMix-UDG (comprises
Taq DNA polymerase (60U/ml), 40mM Tris-HCl (pH8.4), 100mM KCl, 6mMMgCl
2, 400 μ M dGTP, 400 μ M dATP, 400 μ M dCTP, 400 μ M dUTP, 40U/ml UDG and
Green I), 0.5 μ l primer (upstream and downstream) (10 μ M), cDNA and water.Use Rotor-Gene
TM3000 PCR in real time monitoring systems increase, and the PCR cycling condition is as follows: 50 ℃ of UCG activation 2 minutes, 95 ℃ 2 minutes, 94 ℃ of 45 circulations in 5 seconds, then 55 ℃ 10 seconds, 72 ℃ 10 seconds, (spacing increased progressively 0.5 ℃ in 10 seconds) is according to monitoring between 55 ℃-95 ℃
Green fluorescence is drawn melting curve.All samples repeat 3 times.Use Rotor-gene6 software by comparing threshold period (C
t) curve measured with the logit analysis of each measured value copy number.Detect the efficient of pcr amplification by the concentration of successive dilution template cDNA, the data of collecting melting curve are estimated the specificity of pcr amplification.The primer that adds β-actin in each specimen comes the difference between calibration samples.Each experiment sample mRNA expression carries out standardization with β-actinmRNA expression.
Western blotting method detection signal pathway
HUVECs after the overnight incubation, adds n β in serum-free medium
2GPI or HSA (500nM and2 μ M) were hatched 60 minutes, and then adding 40ng/ml was hatched 30 minutes for rhVEGF37 ℃.Adding the cold PBS solution 1ml that contains the 100um sodium vanadate reacts with end, sample places on ice and washs with cold PBS, (contains 10mM Tris-HCl, 5% deoxycholic acid with buffer, 5%Triton X-100,250mM NaCl pH7.5,5mM EDTA, the former sodium vanadate of 0.1mM, serpin cocktail (contains 1mM PMSF, 10ug/ml Leupeptin and1ug/ml aprotinin) carry out cracking, cell in hatching 5 minutes, is used ultrasonic treatment on ice more then.Pyrolysis product 4 ℃ with 12000g centrifugal 15 minutes, the proteinic concentration Micro BCA of supernatant
TMProtein Assay Reagent Kit measures.The albumen of 30ug is splined on the nitrocellulose filter that gel after the polyacrylamide gel electrophoresis of 4-12% is transferred to 0.2um.Film after the transfer was with the TBST that contains 2%BSA or 0.5% defatted milk powder incubated at room 1 hour, then with specific one anti-(phospho-ERK1/2, total-ERK1/2, Akt, phospho-Akt (ser473) or phospho-GSK-3 β (ser9)) hatches again, behind the TBST thorough washing, add anti-rabbit igg (1:1000) incubated at room 1 hour of HRP labelling again.Immunoblotting is observed by enhanced chemiluminescence, carries out the shooting of X line with high performance chemiluminescence film.With Bio-Rad Gel Doc2000 analytical system the density of film image band is measured.Calculate the relative density of the ratio of the density value of each purpose band and the β-actin as measured target protein.
Statistical analysis
Data are represented with mean ± standard deviation.Relatively adopt student ' s t check between two groups, relatively adopt one factor analysis of variance between group, relatively adopt Tukey ' s check between group in twos.There is significant difference p＜0.05 for difference.
The result
β
2GPI is the propagation of the inhibition VEGF and the inductive HUVEC of bFGF of dose dependent
Angiogenesis comprises the partial propagation of endotheliocyte.HUVEC is hatched under different conditions: the β natural, fracture, that the domain deletion form suddenlys change
2GPI (DI-IV and DII-V) or HSA (10nM, 100nM and1 μ M) apply rhVEGF or rhbFGF (10ng/ml) stimulating factor simultaneously, observe β with this
2Whether GPI can influence VEGF and the inductive cell proliferation of bFGF.Endotheliocyte number with MTS method quantitative assay survival.Compare 10nM, 100nM and1000nM n β with BSA
2GPI can be the propagation of the inductive endotheliocyte of inhibition 10ng/ml rhVEGF of dose dependent, its suppression ratio is respectively 11.49 ± 14.31% (mean ± SE, n=6) (P〉0.05), 38.70 ± 8.808% (mean ± SE, n=6) (P＜0.01) and 68.89 ± 6.118% (mean ± SE, n=6) (P＜0.01).In addition, 100nM and1000nM c β
2GPI can suppress the propagation of the inductive endotheliocyte of rhVEGF, its suppression ratio is respectively 22.27 ± 12.76% (mean ± S, n=6) (P〉0.05) and 34.44 ± 8.550% (mean ± SE, n=3) (P＜0.05).DI-IV (100nM and1 μ M) also can suppress the propagation of the inductive endotheliocyte of rhVEGF, and its suppression ratio is respectively 21.32 ± 8.407% (mean ± SE, n=6) (P＜0.05) and 30.32 ± 11.01% (mean ± SE, n=6) (P＜0.05).(see figure 1) on the other hand, n β
2GPI, c β
2GPI and DI-IV can be the propagation of the inductive endotheliocyte of inhibition rhbFGF of dose dependent equally.(see figure 2) and DII-V are for the not influence of propagation of rhVEGF or the inductive endotheliocyte of rhbFGF.
N β
2GPI, c β
2GPI and DI-IV are the migration of the inhibition VEGF and the inductive HUVEC of bFGF of dose dependent
The migration that endotheliocyte passes basement membrane is a committed step of setting up new vessels.In order to observe β
2GPI is in external effect for rhVEGF and the inductive endothelial cell migration of rhbFGF, we have adopted following method: the monolayer endothelial cell that merges is carried out cut form an exposed area to remove a part of cell monolayer, apply intervention then: contrast buffer, rhVEGF (50ng/ml) or rhbFGF (20ng/ml) add n β simultaneously
2GPI or HSA (100nM and1 μ M) were hatched 18 hours.The every porocyte of microscopic examination after 18 hours, we can see with BSA and compare n β in Fig. 3
2GPI can be the inductive cell migration of inhibition rhVEGF (the 100nM n β of dose dependent
2GPI, 37.24 ± 15.75% (mean ± SE, n=6) (P＜0.05); 1000nM n β
2GPI, 83.46 ± 2.589% (mean ± SE, n=6) (P＜0.01)).The result is similar therewith, n β
2GPI also can be the inductive cell migration of inhibition rhFGF of dose dependent and (compare 100nM n β with BSA
2GPI, 38.15 ± 19.89% (mean ± SE, n=6) (P＜0.05); 1000nM n β 2GPI, 76.15 ± 9.350% (mean ± SE, n=6) (P＜0.01)).We can see in Fig. 4,100Nm c β
2GPI and DI-IV can suppress the inductive cell migration of rhVEGF and (be respectively 54.90 ± 6.867% (mean ± SE, n=6) (P＜0.01) and 44.11 ± 11.55% (mean ± SE, n=6) (P＜0.05)), 100nM c β
2The β of GPI and sudden change
2GPI (DI-IV) can suppress the inductive cell migration of rhFGF equally and (be respectively 59.56 ± 4.683% (mean ± SE, n=6) (P＜0.01), 44.06 ± 12.77% (mean ± SE, n=6) (P＜0.01)).In addition, n β
2GPI, c β
2The β of GPI, sudden change
2There is not significant difference for the effect that suppresses cell migration between the GPI (DI-IV).Yet, the β of sudden change
2GPI (DII-V) can not suppress rhVEGF or the inductive cell migration of rhbFGF, and this contains the important sequence of mediation angiogenesis effect with regard to description architecture territory I.
N β
2GPI, c β
2GPI and DI-IV can suppress VEGF and the inductive HUVEC of bFGF on Matri glue blood vessel takes place.
Angiogenesis is a key character with the newborn pipe of endotheliocyte spline structure, so we adopt vascularization to test to study nP β
2The influence that GPI generates for HUVEC blood capillary spline structure.Give on the Matri glue stimulate with rhVEGF or hbFGF (10ng/ml) after, endotheliocyte is gathered into pencil, begins to form pipe spline structure (Fig. 5) after 6 hours.N β
2GPI, c β
2The inhibition rhVEGF that GPI and DI-IV (100nM and1000nM) all can significances and the formation (P＜0.01) (seeing Fig. 6, Fig. 7) of the inductive endotheliocyte pipe of rhbFGF spline structure yet, the β of sudden change
2GPI (DII-V) can not suppress the formation (P〉0.05) of rhVEGF and the inductive endotheliocyte pipe of rhbFGF spline structure.What is interesting is that n β 2GPI only just can suppress the formation of the inductive pipe spline structure of bFGF when concentration is 1000 nM, and n β
2GPI, c β
2GPI and DI-IV all can become the formation (seeing Fig. 6, Fig. 7) of the inhibition rhVEGF and the inductive endotheliocyte pipe of the rhbFGF spline structure of dose dependent.
β
2The mRNA that GPI can suppress rhVEGF and the inductive endotheliocyte KDR/Flk-1 of rhbFGF expresses the then not influence for Flt-1
We study n β at this
2The expression that whether GPI can suppress KDR/Flk-1---receptor that plays a significant role in endothelial cell proliferation, migration and blood vessel take place---.The n β of 500nM and2000nM
2GPI can suppress the expression of the inductive endotheliocyte KDR/Flk-1mRNA of rhVEGF (40ng/ml), can suppress 58.94 ± 14.24% (mean ± SE respectively, n=3) (P〉0.05) and 81.69 ± 5.634% (mean ± SE, n=4) (P＜0.05) sees Fig. 8; It equally also can suppress the expression of the inductive endotheliocyte KDR/Flk-1mRNA of bFGF (40ng/ml), can suppress 92.72 ± 2.270% (mean ± SE, n=3) (P＜0.01) and82.44 ± 7.691% (mean ± SE, n=3) (P＜0.01) respectively.In contrast, n β
2GPI can not suppress the expression of rhVEGF and the inductive endotheliocyte Flt-1 of rhbFGF.
β 2GPI can block rhVEGF and the inductive downstream signal pathway of rhbFGF
Can the interior a series of kinases of activating cell after the receptors bind on rhVEGF and rhbFGF and the endotheliocyte.The existence of endotheliocyte depends on the activation of PI3K, then increases phosphatidylinositols 3,4, and 5 and activate molecule in the various kinds of cell, such as Akt and downstream albumen GSK3 β thereof.RhVEGF and rhbFGF play a role via MAPK approach and then activation Erk-1/2 by the albumen that activates in these paths.In order to study n β
2GPI is for the effect of the downstream signal transduction molecule of rhVEGF and the inductive KDR/Flk-1 triggering of rhbFGF, and we detect ERK1/2, Akt, the activation situation of and GSK3 β.Endotheliocyte is by the n β of 500nM and 2 μ M
2GPI pretreatment 1 hour, then giving with rhVEGF or rhbFGF (40ng/ml) stimulated 30 minutes.Specific antibody with phosphorylation detects ERK1/2, the activation situation of Akt (Ser473) andGSK-3 β (Ser9) (seeing Fig. 9, Figure 10) with western blotting method.N β
2GPI can be the inhibition rhVEGF and the inductive Akt of rhbFGF (Ser473) of dose dependent, the phosphorylation of GSK-3 β (Ser9) and ERK1/2.(seeing Figure 11, Figure 12, Figure 13)
Discuss
Our experimental result shows n β
2GPI, c β
2GPI and DI-IV can be in vitro inhibition rhVEGF-and the inductive endothelial cell proliferation of rhbFGF, migration and vascularization, and this process is regulated by KDR/Flk-1 and downstream signal pathway (comprising MAPK/ERK and PI3K/Akt/GSK3 β path) thereof.
β
2GPI is that a kind of plasma protein of phospholipids incorporate is made up of 5 domains.β
2The prompting of the structure of GPI is inserted lipid membrane at the 5th domain, and two domains (III and IV) prevent Proteolytic enzyme by glycosylation, and two domains (I and II) stretch out from the lipoid surface in addition, so just can with other protein and/or antibodies.Domain V is several different with other, and it contains positively charged lysine and a hydrophobic ring-type C-terminal.The C-terminal of the 5th domain exposes to the surface, therefore at Lys
317With Thr
318Be easy to by plasmin and FXIa hydrolysis, form breaking type β
2GPI.In being arranged, patient's blood plasma of thrombosis and DIC, leukemia, lupus erythematosus can detect breaking type β
2GPI, it is the activated sensitivity label thing of body inner fibrin dissolution.The activation of DIC and fibrinolytic effect is the common pathological changes of tumor patient.The recently many proteolytic fragments of many inhibition angiogenesis or domains that some albumen is hidden discovered, as angiostatin, endostatin, thrombospondin, tissue inhibitor of metalloproteinase can be blocked tumor and change to the angiogenesis phenotype.
In the past studies show that VEGF and VEGFRs are the main regulatory factors of endothelial cell proliferation, migration, VEGF by with its specific receptor interaction inducing endothelial cell angiogenesis.β up to now
2GPI does not illustrate as yet fully for the molecules mechanism that suppresses the inductive angiogenesis of VEGF.Do not have bibliographical information at present about β
2GPI is for the influence of VEGFR and downstream signal pathway developed by molecule thereof.Therefore we are in vitro study n β
2GPI, c β
2The β of GPI and domain deletion mutation
2(DI-IV is DII-V) for the influence of the inductive angiogenesis of VEGF for GPI.
Our result is presented at external, n β
2GPI, c β
2GPI and DI-IV have the effect of powerful angiogenesis inhibitor.N β
2GPI, c β
2GPI and DI-IV can be the inductive endotheliocyte of the VEGF process relevant with angiogenesis that influence of dose dependent, comprise the generation of endothelial cell proliferation, migration and pipe spline structure.
β
2The domain I of GPI mediates its inhibitory action that takes place for the blood vessel of VEGF and the inductive endotheliocyte of bFGF, and β
2The domain V of GPI there is no this effect.What is interesting is anti-β
2The antigenic determinant of the domain I that the antibody of GPI is can facedown relevant with the APS clinical manifestation.APS patient's antibody can be the expression of inducing mononuclear cell VEGF and Flt-1 of dose dependent by the MAPK approach.Present a kind of so new probability, the β of having researched and proposed
2The effect of the angiogenesis inhibitor of the domain I of GPI may be eliminated at the antibody of domain I in APS patient's body.
We suppose β
2The effect of the angiogenesis inhibitor of GPI becomes specific relevant with the VEGF/KDR/Flk-1 system.For verify this hypothesis, our first Application powerful the causing of another one angiogenesis factor---bFGF has studied β
2GPI finds β for the influence of endothelial cell proliferation, migration and angiogenesis
2GPI can suppress the angiogenesis of the inductive endotheliocyte of bFGF equally.These results show β
2The activity of GPI angiogenesis inhibitor is not limited to certain specific part or receptor.
In order to confirm β
2GPI realizes the effect of angiogenesis inhibitor by downward modulation VEGFR, and we have measured Flt-1 and KDR/Flk-1 expression of gene.The real-time quantitative PCR analysis shows β
2GPI can be the inductive KDR/Flk-1 gene expression of inhibition VEGF of dose dependent, and the Flt-1 expression is uninfluenced.KDR/Flk-1 is the main medium that endothelial cell VEGF plays a role.What is interesting is β
2GPI equally also can suppress the inductive KDR/Flk-1 gene expression of bFGF.This just illustrates that bFGF comes induction of vascular new life by indirect activation VEGF/VEGFR path, rather than plays a role by autoreceptor.Previous scholars prompting bFGF makes endotheliocyte more responsive for the stimulation of the VEGF of low concentration by the number of regulating vegf receptor, and our experiment has further confirmed this hypothesis.Therefore, we have reason to believe, are β for the inhibitory action of KDR/Flk-1
2GPI suppresses the main mechanism of VEGF and bFGF inducing endothelial cell angiogenesis function.
Former studies shows that VEGF comes the propagation of inducing endothelial cell and by activating PI3K, increases phosphatidylinositols 3,4,5 by activation MAPK/ERK1/2 path, and activation AKt and downstream molecules thereof are kept the existence of cell.The GSK-3 that in the serine/threonine protein synzyme, expresses, it is the main downstream effects original paper of PI3K/Akt path, Proteolytic enzyme by regulating cyclin D1 and the location of subcellular organelle play a role, the Ser9 phosphorylation that its activity can be mediated by Akt and suppressing.Cyclin D1 is the important medium that is transformed to the S phase by the G1 phase in the endothelial cell period, makes the RB protein phosphorylation and inactivation by forming cyclin D1 and CDK4 complex, discharges E2F then and mediates by the G1 phase and transform to the S phase.Therefore we study β
2GPI whether can the inductive KDR/Flk-1 path of blocking VEGF downstream events, as activation ERK1/2 or make Akt and the GSK-3B phosphorylation.We find β
2GPI can the inductive ERK1/2 of blocking VEGF, the phosphorylation of Akt (Ser473).Consistent with former studies, thus various blood vessel originality molecule such as endostatin, capsaicin, Indian yellow can suppress the synthetic of the phosphorylation of RB and DNA by the downward modulation cyclin D1, and we studies show that β
2GPI can significance inhibition rhVEGF or the phosphorylation of the inductive GSK3 β of rhbFGF (Ser9).Therefore we propose β
2GPI may also be accompanied by by suppressing cyclin D1 expression and RB phosphorylation for the blood vessel formation against function with rhVEGF or the inductive endotheliocyte of rhbFGF and make cell rest on the G0/G1 phase of cell cycle.In our experiment, can be easy to find out β
2GPI expresses the phosphorylation of blocking rhVEGF or inductive MAPK/ERK of rhbFGF and PI3K/Akt/GSK3 β passage downstream signal protein by the mRNA of downward modulation endotheliocyte KDR/Flk-1.These experimental results are in conjunction with β
2The molecular structure of GPI will be for further studying β from now on
2Fine prospect has been opened up in the effect of GPI in angiogenesis cutter system really.
Claims (1)
1. the application of V domain deletion mutation type beta 2 glycoprotein I in the preparation anti-angiogenic drugs.
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