CN104928254A - Mesenchymal stem cell for repairing radioactive intestinal epithelial injury by directionally transporting Rspol - Google Patents
Mesenchymal stem cell for repairing radioactive intestinal epithelial injury by directionally transporting Rspol Download PDFInfo
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Abstract
The invention relates to a mesenchymal stem cell for repairing radioactive intestinal epithelial injury by directionally transporting Rspol. A pEGZ-Term/Rspol retrovirus-expression vector is successfully built on the basis of a built radioactive intestinal epithelial injury mouse model; a gene transfection cell strain C3H10/Rspol capable of stably expressing Rspol is obtained by a gene transfevtion method; through preliminary identification, Rspol protein secreted by the gene transfection cell strain has biological activity; then the C3H10/Rspol cell strain is adoptively transferred to the intestinal epithelium radiation injury mouse by using the character of targeting action of Rspol and mesenchymal stem cell on the intestine stem cells for repairing intestinal epithelial injury, so that the repair of the radioactive intestinal epithelial injury can be effectively accelerated.
Description
Technical field
The present invention relates to a kind of for orientation conveying Rspo1 to repair radioactivity enteric epithelium damage mescenchymal stem cell and application, particularly relate to a kind of Rspo1 gene transfection mescenchymal stem cell strain repair vector and repairing the application in enteric epithelium radiation injury, belong to gene engineering technology field.
Background technology
Along with the development of nuclear industry, the widespread use of nuclear science technology and the threat of nuclear terror raid, how giving treatment to acute radiation injury becomes demand urgent now.Enteron aisle is very responsive to ionizing rays, and enteric epithelium structure and function is impaired is that acute radiation venereal disease is difficult to one of key factor of giving treatment to.Intestinal crypts stem cell and stem cell niche subject to severe risks of damage thereof are the basic reasons of intestinal crypts-fine hair regeneration barrier, are the key pathological mechanism of enteric epithelium structure and function radiation injury.
Mescenchymal stem cell (Mesenchymal stem cells, MSCs) be that a group derives from mesoderm, the heterogeneous very strong multipotential stem cell with self-renewal capacity, adipocyte can be divided into, scleroblast, chondrocyte, liver cell, neuronal cell, myocyte, the cells such as epithelial cell.Large quantity research shows, mescenchymal stem cell can chemotactic to the position of injured inflammation, possessing potential immunoregulation capability, anti-inflammatory power and trophism, is organizational project, the ideal tools of regenerative medicine and treatment of autoimmune diseases.
Rspo1 (R-spondin1) belongs to R-spondin (roof plate-specific spondin) family member, it is a kind of shla molecule recently found, be made up of 263 amino-acid residues, containing 1 I type coagulase spline structure territory (thrombospondin type I motif) and 2 furin spline structure territories (furin like domain), be participate in the vertebrate important factor grown, be expressed in the tissues such as enteron aisle, pancreas and suprarenal gland, sexual gland, prostate gland, kidney and brain.Recently find, Rspo1, by regulating gut epithelial stem cells β-catenin activation signals, promotes intestinal crypts stem cells hyperplasia and differentiation, recovers the integrity of intestinal villus and intestinal crypts structure.Give the repair ability that exogenous Rspo1 can improve enteric epithelium, the pathological factors such as Summing Factor active oxygen radical that reduce inflammation are to the damage of intestinal mucosa.And mescenchymal stem cell not only possesses good immune suppression function, the inflammatory damage of local organization can be alleviated, and there is the feature to site of tissue damage migration.In addition, people Rspo1 can act on mouse intestinal crypts stem cell, produces short propagation, differentiation and existence effect.
Therefore, how mescenchymal stem cell is combined with Rspo1 and is applied to the enteric epithelium structure and function brought because of nuclear radiation and damage, become current problem demanding prompt solution.
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide a kind of mescenchymal stem cell damaged to repair radioactivity enteric epithelium for orientation conveying Rspo1, this mescenchymal stem cell, can the directed enteric epithelium repairing rasdiation damage by the immunosuppressive action of C3H10 T1/2 and Rspo1 being urged reproduction restraint effect associating.
Technical scheme of the present invention is:
Repair a construction process for the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1, comprise the steps:
(1) preparation of recombinant retroviral expression vector: design and synthesize primer pair, wherein the sequence of upstream primer is SEQ ID:1, the sequence pair of downstream primer is SEQ ID:2, with people Rspo1 full length cDNA sequence for template, high-fidelity enzyme KOD-neo plus is adopted to obtain PCR primer by pcr amplification, restriction enzyme EcoRI and BamHI is adopted to carry out double digestion to retroviral vector pEGZ-Term and the above-mentioned PCR primer through reclaiming purifying, adopt T4DNA ligase enzyme connect again purified after product, transform competent E. coli TOP10, and select positive bacterium colony, then extracting plasmid, and the plasmid called after pEGZ-Term/Rspo1 that this is extracted, construction obtains recombinant retroviral expression vector pEGZ-Term/Rspo1,
(2) preparation of recombinant retrovirus: by 293T cell with 1 × 10
5/ ml density is inoculated in six orifice plates, when Cell abundance reaches 80% ~ 90%, with lipofection, recombinant plasmid pEGZ-Term/Rspo1, helper viral vector pHIT456 and pHIT60 are imported 293T cell simultaneously, obtain the recombinant retrovirus with infection ability; Cultivation viral supernatants is collected for subsequent use respectively at 48h and 96h;
(3) structure of Rspo1 gene transfection mescenchymal stem cell strain: collection gained viral supernatants 0.5ml in (2) is added the RPM1640 substratum 0.5ml containing 10%FCS, and adds Polybrane, by 2 × 10 by 8 μ g/ml
4/ ml suspendible mouse mesenchymal cell C3H10 T1/2, is placed in 24 well culture plates and cultivates, and after cultivating 48h, digestion C3H10 T1/2 cell also presses 0.5 × 10
4/ ml re-suspended cell, is inoculated in 24 orifice plates by 1ml/ hole, pressurizes screen by 0.5 μ g/ml G418, grows to enlarged culturing enough greatly, called after C3H10/Rspo1 until clone, obtains Rspo1 gene transfection mescenchymal stem cell strain C3H10/Rspo1.
Its further technical scheme is:
Described in step (1), upstream primer is with EcoRI restriction enzyme site, and described downstream primer is with BamHI restriction enzyme site and His label.
Described in step (1), pcr amplification condition is: denaturation 95 DEG C, 3min; Sex change 98 DEG C, 10s; Renaturation and extension 68 DEG C, 40s; To increase 30 circulations; Finally extend 68 DEG C, 5min.
The invention also discloses the mescenchymal stem cell C3H10/Rspo1 damaged to repair radioactivity enteric epithelium for orientation conveying Rspo1 that a kind of above-mentioned construction process builds gained.
Its further technical scheme is:
Described C3H10/Rspo1 can stably express people Rspo1 protein molecule, and this people Rspo1 protein molecule target can be migrated to enteron aisle damaged position.
In the supernatant liquor of described C3H10/Rspo1 Rspo1 protein molecule there is biological activity.
The invention also discloses described for orientation conveying Rspo1 to repair the purposes of the mescenchymal stem cell of radioactivity enteric epithelium damage, described C3H10/Rspo1 is for the preparation of target protection and repair the gut epithelium damage medicine or preparation that nuclear radiation causes.
Its further technical scheme is:
The characteristic that mouse mesenchymal cell C3H10 T1/2 can express CXXR4 Chemokine Receptors at directed chemotactic to the application at the enteric epithelium radiation injury position caused by nuclear radiation.
Mouse mesenchymal cell C3H10 T1/2 can express the cytokine storm of characteristic after suppressing radiation injury and the application of secondary response of PD-L1, B7H3, B7H4, TGF-β negativity membrane molecule and secretion TGF-β, IL-10 and PGE2 immunosuppression molecule.
People Rspo1 protein molecule is promoting the application in intestinal crypts stem cells hyperplasia and differentiation.
By such scheme, the present invention at least has the following advantages: the present invention utilizes enteric epithelium irradiated mice model, by the immunosuppressive action of mouse mesenchymal cell C3H10 T1/2 and people Rspo1 molecule being urged reproduction restraint effect associating, be used for the directed enteric epithelium repairing rasdiation damage, for prepare acute radiation sickness process in repair the medicine of enteric epithelium structure and function or preparation provides good basis.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the survival curve of different irradiation dose mouse;
Fig. 2 is mouse sign change after irradiation;
Fig. 3 is Mouse Weight change after irradiation;
Fig. 4 is the change of the little broiler chick of different number of days after irradiation;
Fig. 5 is the change of different number of days small intestinal length after irradiation;
Fig. 6 is change HE dyeing 10 × and the 20 × enlarged view of different number of days small intestine pathomorphism after irradiation;
Fig. 7 is that after irradiation, different number of days intestinal villi length compares schematic diagram with crypts number;
Fig. 8 is Lgr5 in different number of days crypts of small intestine after irradiation
+the change of stem cell;
Fig. 9 is the comparison of different number of days intestinal crypt stem cells quantity after irradiation;
Figure 10 is the structure of recombinant expression vector Pegz-Term/Rspo1, wherein M:DL2000 DNA marker; 1:pEGZ-Term/Rspo1 recombinant vectors; 2:pEGZ-Term/Rspo1 recombinant vectors adopts the product after EcoRI and BamHI double digestion; The product of 3: people Rspo1 full-length gene after pcr amplification;
Figure 11 is the expression that RT-PCR detects Rspo1 mRNA in C3H10/Rspo1 cell, wherein A:M:DL2000 DNA marker; 1:pEGZ-Term/Rspo1 recombinant vectors; 2:C3H10/Rspo1; 3:C3H10/mock; 4:C3H10 T1/2; B:M:DL2000 DNA marker; 1:C3H10/Rspo1; 2:C3H10/mock; 3:C3H10 T1/2;
Figure 12 is the result figure of C3H10/Rspo1 cell expressing His-Rspo1 fusion rotein;
Figure 13 is SW480 cell surface expression Rspo1 acceptor Lgr5;
Figure 14 is that C3H10/Rspo1 cell strain supernatant promotes SW480 cell proliferation (* P<0.05, * * P<0.01);
Figure 15 is C3H10 T1/2 cell strain surface expression Chemokine receptor CXCR4;
Figure 16 is C3H10 T1/2 endoglin expression PD-L1, B7H3, B7H4 and TGF-β, and wherein empty body peak is negative peak, and entity peak is detected peaks;
Figure 17 is the expression of IL-4, IL-10 and IFN-γ in C3H10 T1/2 cell, and wherein empty body peak is negative peak, and entity peak is detected peaks;
Figure 18 is C3H10 T1/2 emiocytosis PGE2;
Figure 19 is the survival curve of different Transplanted cells group mouse;
Figure 20 is the body weight change of different Transplanted cells group mouse;
Figure 21 is the sign change of mouse after the reparation of enteric epithelium radiation damage.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
One, the foundation of enteric epithelium irradiated mice model and injuring rule
Adopt in the present invention 8-10 week age Lgr5-EGFP-ires-CreERT2 transgenic male mice (Jackson Laboratories, Bar Harbor, Maine) set up enteric epithelium irradiated mice model.Above-mentioned mouse is divided into 10Gy, 12Gy, 14Gy, 16Gy and 18Gy group according to irradiation dose difference, often organizes 5 mouse.By experimental mice with after 1% Sodital intraperitoneal anesthesia, four limbs stretch, and are fixed on irradiation table with adhesive tape.Irradiate belly merely through linear accelerator single, dose rate is 200mu/min, and irradiation dose divides 10Gy, 12Gy, 14Gy, 16Gy and 18Gy respectively.Observe the mental status of each group of mouse, survival time, determine minimum lethal dose.Its result is shown in Figure 1, and as can be seen from Figure 1, the mouse 5d of 18Gy irradiation dose irradiation group starts to occur death, and 9d mouse is all dead.Mouse all energy long-term survivings of other dose delivery group, during 30d, survival rate is still 100%.Therefore 18Gy irradiation dose is adopted to be the minimum lethal dose setting up radiativity enteric epithelium damage mouse model in the present embodiment.
The observation of its pathological change after radiation is carried out to mouse as described below:
The change of body weight and sign.After 18Gy irradiation dose irradiates mouse web portion merely, from 0d, observe Mouse Weight every day and sign changes, measure the body weight of mouse, observe the situation of ingesting of mouse, sign change and survival condition, until dead mouse.Namely present result display mouse 3.5d after irradiation hair color obfuscation, appetite poorly, movable reduce, slow in reacting and with symptoms such as bloody stools, extend symptom further serious (Fig. 2) in time; Continue discovery of weighing in, compared with normal healthy controls group, along with the increase of irradiation number of days, Mouse Weight progressively alleviates, and after irradiation 3d, body weight change difference has statistical significance (Fig. 3).
Intestinal tract injury gross examination of skeletal muscle.Observe intestinal crypts-fluff structures impaired: respectively at 0.5d, 3.5d and 6d after irradiation, cervical dislocation puts to death mouse, measures mouse intestinal length, and observe intestines wall hemorrhagic and change.As shown in Figure 4,18Gy irradiation dose irradiates merely 3.5d after mouse web portion, and mouse small intestine radiation injury is serious, and small intestine enteron aisle manifests the hemorrhage and rotten to the corn symptom of mucous membrane dispersivity, and along with the increase of irradiation number of days, edematous condition appears in enteron aisle, and hemorrhage and rotten to the corn situation is aggravated.6d after irradiation, mouse intestinal occurs that enteron aisle paralysis, intestines wall are obviously thinning, even transparence, and with intestines wall hemorrhage (Fig. 5 C).Compared with normal healthy controls group, 3.5d and 6d after irradiation, the length of small intestine obviously shortens, and difference has statistical significance (Fig. 5 (A, B)).
The measurement of small intestinal villous height.Respectively at 0.5d, 3.5d and 6d after irradiation, cervical dislocation puts to death mouse, get intestinal segment under duodenum Qu Shi ligament, PBS liquid rinses intestinal lumen contents well, fix with 4%PFA, then dewater successively with 5%, 10%, 20%, 30% sucrose, OCT embedding after carry out tissue slice, adopt conventional H E dyeing, the intestinal villus that random selecting 10 root architecture is complete under the microscope of band ocular micrometer, measures height of naps.
As shown in Figure 6, irradiation 3.5d, small intestine epithelium suffers radiation injury, and intestinal villi shortens, and rupture in top, come off and occur erosion, epithelial cell is downright bad in a large number, loses normal morphology, and crypts distortion bottom small intestine, number reduces.Shining rear 6d, fine hair-crypts structure havoc, fine hair arrangement is loose, and crypts loses intact form, and number obviously reduces.Found out by Fig. 7 A, according to rear 3.5d and 6d, villus length is 27.90 ± 4.42 μm and 19.31 ± 6.25 μm, and normal healthy controls group mean length is 42.79 ± 4.99 μm and 42.06 ± 5.08 μm, obviously shortens according to rear villus length.When being found out according to rear 3.5d and 6d by Fig. 7 B, in the visual field, the number of complete crypts is 7.90 ± 1.00 and 6.60 ± 1.96, normal healthy controls group number is 18.9 ± 2.23 and 19.2 ± 3.55, and after irradiation, crypts number obviously reduces, and difference has statistical significance.
Crypts number is measured and Lgr5 in intestinal crypts
+stem cell population changes.The tissue slice of above-mentioned HE is placed in low power opticmicroscope, observes the integrity of crypts in intestinal tube, and the complete crypts number under adding up the different visual field, often organize statistics 10 visuals field, average; Intestinal tissue section DAPI is contaminated core 30s; PBS cleans 3 times, each 5min; Add anti-cancellation fluid-tight sheet, under being placed in laser confocal microscope, observe Lgr5 in crypts
+cell, and add up Lgr5 in the caudal recessus of the different visuals field
+the number of cell, often organizes statistics 10 visuals field, averages.Intestinal crypts stem cell expresses Lgr5, and Lgr5-EGFP-ires-CreERT2 transgenic mice is chosen in this experiment, with GFP (green fluorescent protein) spike Lgr5
+intestinal crypts stem cell.Confocal microscope is observed and is irradiated rear 0.5d, 3.5d and 6d crypts of small intestine position Lgr5
+intestinal crypts stem cell, as shown in Figure 8, green fluorescence is Lgr5
+intestinal crypts stem cell expresses mark.Shining rear 3.5d, in intestinal crypts, Crypt Stem Cells quantity reduces in a large number; Shining rear 6d, intestinal crypts stem cell disappears and the subject to severe risks of damage of crypts form.Compared with normal healthy controls group, be obviously less than untreated fish group according to rear 3.5d and 6d intestinal crypts stem cell population, difference has statistical significance (Fig. 9).
Above-mentioned all data represent with mean ± standard deviation (Mean ± SD), process with SPSS 13.0 software, and assembly compares employing t inspection.P<0.05 is that difference has statistical significance.
That is: the minimum lethal dose of simple abdominal irradiation mouse is 18Gy.After mouse web portion is subjected to 18Gy irradiation, mouse activity reduces, weight loss is with symptoms such as bloody stools, along with the prolongation of time after irradiation, the length of small intestine obviously shortens with rotten to the corn and ulcer, pathological section result shows: after irradiation, intestinal villi top is obviously rotten to the corn, comes off, and villus length obviously shortens; Crypts damaged deformation, inner necrocytosis, complete crypts number obviously reduces; Lgr5 in intestinal crypts
+reduce rapidly in the stem cell short period of time.
Two, the structure of Rspo1 gene transfection mescenchymal stem cell strain and identification of its biological activity
1, the structure of recombinant retroviral expression vector
According to people Rspo1 full length cDNA sequence (Genebank ID:BC114966) and His sequence label, design and synthesize the upstream primer with EcoRI restriction enzyme site:
EcoRI-F:5’-ATTAGAATTCATGCGGCTTGGGCTGTGTGTGG-3’(SEQ ID：1)
With the downstream primer with BamHI restriction enzyme site and His label:
BamHI-R:5’-GTGTGGATCCTTAATGGTGATGGTGATGATGGGCAGGCCCTGCAGATGTG-3’(SEQ ID：2)
Adopt high-fidelity enzyme KOD-neo plus (being purchased from Toyobo, Japan) by pcr amplification, PCR condition is: denaturation 95 DEG C, 3min; Sex change 98 DEG C, 10s; Renaturation and extension 68 DEG C, 40s; To increase 30 circulations; Finally extend 68 DEG C, 5min.
Reclaim PCR primer, with restriction enzyme EcoRI and BamHI (being purchased from Canadian Fermentas company), double digestion is carried out to recovery purified product and retroviral vector pEGZ-Term, T4 ligase enzyme (being purchased from Japanese TaKaRa company) connects product after purification again, transform competent E. coli TOP10 (being purchased from Novagen company of the U.S.).Selecting positive bacterium colony, verifying through boiling bacterium PCR, double digestion checking and DNA sequencing identify that correct clone is for amplification, extracting plasmid (plasmid extraction test kit: be purchased from Hangzhou Axyen company), and called after pEGZ-Term/Rspo1.Adopt double digestion method the result as shown in Figure 10, result display recombinant plasmid can discharge fragment of the same size with Rspo1 full-length gene through EcoRI and BamHI double digestion, and shows that in recombinant vectors, Rspo1 gene order is correct through two-way DNA sequencing.
2, the preparation of recombinant retrovirus and the structure of gene transfecting cell strain C3H10/Rspo1 and C3H10/mock
By 293T cell (being purchased from ATCC company of the U.S.) with 1 × 10
5/ ml density is inoculated in six orifice plates, when Cell abundance reaches 80% ~ 90%, with lipofection, recombinant plasmid pEGZ-Term/Rspo1, helper viral vector pHIT456 and pHIT60 are imported 293T cell simultaneously, obtain the retrovirus with infection ability; For subsequent use respectively at 48h and 96h collection culture supernatant, prepare the recombinant retrovirus supernatant with infection ability.Infect 293T cell with empty carrier pEGZ-Term after the same method simultaneously, and collect viral supernatants.Then viral supernatants 0.5ml is added the RPM1640 substratum 0.5ml containing 10%FCS, and add Polybrane (being purchased from Sigma Co., USA), by 2 × 10 by 8 μ g/ml
4/ ml suspendible C3H10 T1/2 cell, is placed in 24 well culture plates and cultivates.After cultivating 48h, digestion C3H10 T1/2 cell also presses 0.5 × 10
4/ ml re-suspended cell, is inoculated in 24 orifice plates by 1ml/ hole, pressurizes screen by 0.5 μ g/ml G418, grows to enlarged culturing enough greatly, called after C3H10/Rspo1 until clone.Prepare negative control C3H10/mock cell strain in the same way simultaneously.
3, the Rspo1 mrna expression of gene transfecting cell strain C3H10/Rspo1 is detected by RT-PCR method, its specific practice is: with each cell strain total serum IgE of Trizol (being purchased from American I nvitroGen company) extracting and reverse transcription is cDNA, respectively with the cDNA of C3H10 T1/2, C3H10/mock, C3H10/Rspo1 tri-kinds of cells for template amplification Rspo1 gene fragment.
Rspo1 amplification upstream primer is: 5 '-CTGGAGAGGAACGACATCCG-3 ', (SEQ ID:3)
Downstream primer is: 5 '-GGTCTCCTTGGTGTCAGAGC-3 ', (SEQ ID:4)
Expection product length is 398bp;
β-actin amplification upstream primer is: 5 '-ATCTGGCACCACACCTTCTACA-3 ', (SEQ ID:5)
Downstream primer is: 5 '-GATAGCACAGCCTGGATAGCAA-3 ', (SEQ ID:6)
Expection product length is 140bp.
Result display obtains the gene fragment of expection size for template can increase with C3H10/Rspo1 cell strain cDNA, and parental cells C3H10 T1/2 and empty vector control cell C3H10/mock produces (Figure 11) without specific band.
4, the expression of His labelled protein in Flow cytometry gene transfecting cell strain born of the same parents is adopted
Its specific practice is: the C3H10 T1/2 in vegetative period that takes the logarithm (should be mouse mesenchymal cell strain, and be purchased from Shanghai Chinese Academy of Sciences cell bank), C3H10/mock, C3H10/Rspo1 cell, by 5 × 10
5/ test, with PBS buffer solution 1 time, add stationary liquid 200 μ l/test fixed cell 30min, with PBS buffer solution 1 time, add 200 μ l/test perforation liquid effect 30min, add the straight labeling antibody of Anti-His-PE (being purchased from German Miltenyi company) and carry out dyeing in born of the same parents, normal temperature hatches 30min, perforation liquid washs 1 time, adopts the expression of His-Rspo1 fusion rotein in flow cytomery born of the same parents.Result shows, C3H10/Rspo1 high expression level His-Rspo1 fusion rotein (Figure 12), and does not detect that His tag fusion protein is expressed in C3H10 T1/2 and C3H10/mock cell.
5, the expression of Flow cytometry SW480 cell surface Rspo1 acceptor Lgr5 albumen and cell counting detect gene transfecting cell strain supernatant to the impact of colon cancer cell line SW480 multiplication capacity
Its specific practice is: SW480 cell in vegetative period of taking the logarithm (be colon cancer cell line, be purchased from ATCC company of the U.S.), by 1 × 10
5individual/test adds the anti-human Lgr5 of rabbit many anti-(being purchased from American AB GENT company), 5 μ l/test, hatch 45min for 4 DEG C, PBS washing once, add the donkey anti-rabbit fluorescence two anti-(being purchased from American AB GENT company) of PE mark, 5 μ l/test, hatch 15min for 4 DEG C, PBS washes twice, and adopts the expression of Lgr5 molecule on flow cytomery cell surface.Logarithmic phase C3H10 T1/2, C3H10/mock, C3H10/Rspo1 cell culture medium is changed to serum free medium and cultivates collecting cell supernatant after 48h, simultaneously using serum free medium as negative control group, Rspo1 albumen (10ng/ml) is as positive controls, be laid in 96 orifice plates by above-mentioned 5 groups of supernatants with 50 μ l/ holes respectively, often group establishes 3 multiple holes.By the enzymic digestion of SW480 cell tryptase, be resuspended in 1640 substratum containing 10%FCS, adjustment cell concn is 1 × 10
5/ ml, is inoculated in 96 orifice plates with 50 μ l/ holes, 5%CO
2, cultivate in 37 DEG C of incubators.Cell counting is adopted to detect cell proliferative condition in 24h, 48h and 72h.
Detected result shows: SW480 cell surface high expression level Rspo1 acceptor Lgr5 albumen (Figure 13), utilize cell counting to detect each group of supernatant to find the impact of SW480 ability of cell proliferation: compared with C3H10/mock supernatant cleer and peaceful on substratum, C3H10 T1/2, the upper cleer and peaceful Rspo1 albumen of C3H10/Rspo1 obviously promotes SW480 cell proliferation (Figure 14)
All data represent with mean ± standard deviation (Mean ± SD) above, process with SPSS 13.0 software, and assembly compares employing t inspection.P<0.05 is that difference has statistical significance.
The present embodiment adopts mescenchymal stem cell strain C3H10 T1/2 as parental cells, successfully construct the gene transfecting cell strain C3H10/Rspo1 of stably express people Rspo1 molecule, in its supernatant, Rspo1 albumen has biologic activity, for important basic substance has been established in the research of target intestinal stem cell treatment enteric epithelium damage.
Three, the radiation injury of mouse enteric epithelium is repaired in the strain of Rspo1 gene transfection mescenchymal stem cell
1, the expression of Flow cytometry C3H10 T1/2 cell strain surface chemokine receptor CXCR4
Its specific practice is: C3H10 T1/2 cell 5 × 10 in vegetative period of taking the logarithm
5/ test, with the PBS buffer solution 1 time containing 0.3%FCS, add CXCR4mAb-FITC antibody staining, hatch 30min for 4 DEG C, PBS buffer solution 2 times, the expression of Flow cytometry cell surface CXCR4 molecule, detected result is as shown in figure 15, C3H10 T1/2 cell surface expression CXCR4, illustrate C3H10 T1/2 cells i injection after can directional migration to damage location.
2, the expression of Flow cytometry C3H10 T1/2 cell strain surface PD-L1, B7H3, B7H4 and TGF-β, and the expression of IL-4, IL-10 and IFN-γ in C3H10 T1/2 cell strain
Its specific practice is: C3H10 T1/2 cell 5 × 10 in vegetative period of taking the logarithm
5/ test, with the PBS buffer solution 1 time containing 0.3%FCS, add 5 μ l PD-L1, B7H3, B7H4 and TGF-β monoclonal antibody (purchased from American eBioscience company) dyeing respectively, hatch 30min for 4 DEG C, PBS buffer solution 2 times, the expression of Flow cytometry cell surface PD-L1, B7H3, B7H4, TGF-β tetra-Middle molecule, detected result is see Figure 16.
To take the logarithm C3H10 T1/2 cell 5 × 10 in vegetative period
5/ test, with PBS buffer solution 1 time, add stationary liquid 200 μ l/test fixed cell 30min, with PBS buffer solution 1 time, add 200 μ l/test perforation liquid effect 30min, add IL-4, IL-10 and IFN-γ monoclonal antibody ((purchased from American eBioscience company)) respectively and carry out dyeing in born of the same parents, normal temperature hatches 30min, perforation liquid washs 1 time, adopts the expression of IL-4, IL-10 and IFN-γ in flow cytomery born of the same parents.
Result shows, and C3H10 T1/2 cytolemma all expresses negativity costimulatory molecules PD-L1, B7H3 and B7H4 (Figure 16); By intracellular cytokine dyeing and the display of Flow cytometry result, C3H10 T1/2 cell can produce negative cells factor TGF-β (Figure 16), IL-4 and IL-10 (Figure 17), and does not produce IFN-γ (Figure 17).
3, ELISA method detects the content of PGE2 in C3H10 T1/2 cell conditioned medium
Its specific practice is: adjustment C3H10 T1/2 cell concn is 5 × 10
4/ ml, is inoculated in 6 orifice plates with 2ml/ hole, 5%CO
2, cultivate in 37 DEG C of incubators, respectively at 1d, 2d and 3d collecting cell supernatant, adopt the content of PGE2 in the PGE2ELISA kit detection cell supernatant of R & D company, its result is see Figure 18.As seen from Figure 18, C3H10 T1/2 cell can secrete PGE2, and first three sky cultivated, the content of the PGE2 of its secretion increases gradually.
4, enteric epithelium irradiated mice is repaired in the strain of Rspo1 gene transfection mescenchymal stem cell, and the present embodiment adopts orbital vein to transplant MSCs
Its specific practice is: Lgr5-EGFP-ires-CreERT2 transgenic male mice 30,8-10 week age, mouse is divided into 5 groups: Normal group, PBS control group, C3H10 T1/2 transplantation group, C3H10/mock transplantation group, C3H10/Rspo1 transplantation group; Except Normal group, 18Gy single dose is adopted to irradiate mouse web portion merely.Collect C3H10 T1/2, C3H10/mock, C3H10/Rspo1 cell of logarithmic phase, trysinization, 1200rpm, centrifugal 5min, abandons supernatant; Aseptic PBS washing twice; After irradiation, (1 × 10 is implanted in Mice Body immediately with retroorbital venous injection system
6individual/50 μ l/ are only), PBS group injects the aseptic PBS of 50 μ l in the same fashion; After 48h, repeated embryo transfer once.Every day observes body weight and the survival state of often organizing mouse, and carries out statistical analysis to result.
A. different Transplanted cells group is on the impact of enteric epithelium irradiated mice survival rate
After 18Gy irradiation dose irradiates mouse web portion merely, orbital vein injection is adopted to be implanted into respectively in Mice Body at twice in C3H10 T1/2, C3H10/mock, C3H10/Rspo1 cell and PBS.Observe the survival condition often organizing mouse, as shown in figure 19, C3H10 T1/2 transplantation group 6d starts to occur death, and 30d survival rate is 20%; C3H10/mock transplantation group 5d starts to occur death, and 12d is all dead; C3H10/Rspo1 transplantation group 7d starts to occur death, and 30d survival rate is 40%; And PBS control group 5d starts to occur death, 7d is all dead.As can be seen from the survival curve of each group of mouse, the mouse of C3H10/Rspo1 Transplanted cells to enteric epithelium radiation injury has certain repair, and this repair is greater than simple C3H10 T1/2 Transplanted cells.
B. different Transplanted cells group is on the impact of enteric epithelium irradiated mice body weight
As seen from Figure 20,18Gy irradiation dose irradiates intestinal tract injury serious, and initial stage Mouse Weight declines obviously.Wherein, Mouse Weight loses speed: PBS group >C3H10/mock transplantation group >C3H10 T1/2 transplantation group >C3H10/Rspo1 transplantation group, and the Mouse Weight of 9d C3H10 T1/2 transplantation group and C3H10/Rspo1 transplantation group starts slowly to recover after irradiation, and the resume speed of C3H10/Rspo1 transplantation group mouse is faster than C3H10 T1/2 transplantation group.
C. after the reparation of enteric epithelium radiation damage, the sign of mouse changes
C3H10/Rspo1 cells i is implanted into the mouse suffering x ray irradiation x, the symptoms such as mouse appetite after radiation damage is deteriorated, activity reduces, slow in reacting improve, but mouse has occurred adjoint alphosis by x ray irradiation x position, may be owing to causing mouse irradiation position hair follicles damage (Figure 21) after irradiation.
All data represent with mean ± standard deviation (Mean ± SD) above, process with SPSS 13.0 software, and assembly compares employing t inspection.P<0.05 is that difference has statistical significance.
The present embodiment research confirms that mouse mesenchymal cell strain C3H10 T1/2 expresses CXCR4 molecule, shows that it has possessed the ability to radiation injury position directional migration; Research shows that C3H10 T1/2 expresses the negativity membrane molecules such as PD-L1, B7H3 and B7H4, and produce the immune Negative Factor such as TGF-β, IL-10 and PGE2, this contributes to radiation injury enteric epithelium from the cytokine storm after inflammation damnification and Secondary cases immunologic injury.
The gene transfecting cell strain C3H10/Rspo1 of the stably express people Rspo1 molecule constructed by the present invention, can secrete Rspo1 albumen and its target is migrated to radiation injury position, promotes propagation and the activation of intestinal stem cell.The present embodiment have studied the impact that different transplantation group is repaired radiativity enteric epithelium damage mouse, finds that simple C3H10 T1/2 cell of transplanting has certain repair to the mouse that radiativity enteric epithelium damages; And transplant C3H10/Rspo1 cell; the immunosuppressive action protection damaged intestine stem cell of C3H10 T1/2 cell can not only be played; also by secretion Rspo1 protein activation Wnt signal; promote propagation and the activation of intestinal stem cell; both combine jointly, and reparation is by the enteric epithelium of radiation damage, and this may be the inherent mechanism that this experimental mice survival rate is high.
Protection intestinal crypts stem cell and stem cell niche, the stem cells hyperplasia of promotion intestinal crypts and differentiation is indispensable during radioactivity enteric epithelium is repaired and two of interdependence large key elements again.The present invention utilizes enteric epithelium irradiated mice model, on the basis inquiring into radiation skin damage pathomechanism on intestines, by the immunosuppressive action of C3H10 T1/2 and Rspo1 being urged reproduction restraint effect associating, and the directed enteric epithelium repairing rasdiation damage, this may be the novel available strategy of repairing enteric epithelium structure and function in the process for the treatment of acute radiation sickness.
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (10)
1. repair a construction process for the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1, it is characterized in that: comprise the steps:
(1) preparation of recombinant retroviral expression vector: design and synthesize primer pair, wherein the sequence of upstream primer is SEQ ID:1, the sequence pair of downstream primer is SEQ ID:2, with people Rspo1 full length cDNA sequence for template, high-fidelity enzyme KOD-neo plus is adopted to obtain PCR primer by pcr amplification, restriction enzyme EcoRI and BamHI is adopted to carry out double digestion to retroviral vector pEGZ-Term and the above-mentioned PCR primer through reclaiming purifying, adopt T4DNA ligase enzyme connect again purified after product, transform competent E. coli TOP10, and select positive bacterium colony, then extracting plasmid, and the plasmid called after pEGZ-Term/Rspo1 that this is extracted, construction obtains recombinant retroviral expression vector pEGZ-Term/Rspo1,
(2) preparation of recombinant retrovirus: by 293T cell with 1 × 10
5/ ml density is inoculated in six orifice plates, when Cell abundance reaches 80% ~ 90%, with lipofection, recombinant plasmid pEGZ-Term/Rspo1, helper viral vector pHIT456 and pHIT60 are imported 293T cell simultaneously, obtain the recombinant retrovirus with infection ability; Cultivation viral supernatants is collected for subsequent use respectively at 48h and 96h;
(3) structure of Rspo1 gene transfection mescenchymal stem cell strain: collection gained viral supernatants 0.5ml in (2) is added the RPM1640 substratum 0.5ml containing 10%FCS, and adds Polybrane, by 2 × 10 by 8 μ g/ml
4/ ml suspendible mouse mesenchymal cell C3H10T1/2, is placed in 24 well culture plates and cultivates, and after cultivating 48h, digestion C3H10T1/2 cell also presses 0.5 × 10
4/ ml re-suspended cell, is inoculated in 24 orifice plates by 1ml/ hole, pressurizes screen by 0.5 μ g/ml G418, grows to enlarged culturing enough greatly, called after C3H10/Rspo1 until clone, obtains Rspo1 gene transfection mescenchymal stem cell strain C3H10/Rspo1.
2. the construction process repairing the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1 according to claim 1, it is characterized in that: described in step (1), upstream primer is with EcoRI restriction enzyme site, described downstream primer is with BamHI restriction enzyme site and His label.
3. the construction process repairing the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1 according to claim 1, is characterized in that: described in step (1), pcr amplification condition is: denaturation 95 DEG C, 3min; Sex change 98 DEG C, 10s; Renaturation and extension 68 DEG C, 40s; To increase 30 circulations; Finally extend 68 DEG C, 5min.
4. according to claim arbitrary in claim 1-3, construction process builds the mescenchymal stem cell C3H10/Rspo1 repairing the damage of radioactivity enteric epithelium for orientation conveying Rspo1 of gained.
5. the mescenchymal stem cell repairing the damage of radioactivity enteric epithelium for orientation conveying Rspo1 according to claim 4, it is characterized in that: described C3H10/Rspo1 can stably express people Rspo1 protein molecule, and this people Rspo1 protein molecule target can be migrated to enteron aisle damaged position.
6. according to claim 4 for orientation conveying Rspo1 repair radioactivity enteric epithelium damage mescenchymal stem cell, it is characterized in that: in the supernatant liquor of described C3H10/Rspo1 Rspo1 protein molecule there is biological activity.
7. purposes of repairing the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1 according to claim 4, is characterized in that: the gut epithelium damage medicine that described C3H10/Rspo1 protects for the preparation of target and reparation nuclear radiation causes or preparation.
8. the purposes of mescenchymal stem cell of repairing the damage of radioactivity enteric epithelium for orientation conveying Rspo1 according to claim 7, is characterized in that: the characteristic that mouse mesenchymal cell C3H10T1/2 can express CXXR4 Chemokine Receptors at directed chemotactic to the application at the enteric epithelium radiation injury position caused by nuclear radiation.
9. purposes of repairing the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1 according to claim 7, is characterized in that: mouse mesenchymal cell C3H10T1/2 can express the cytokine storm of characteristic after suppressing radiation injury and the application of secondary response of PD-L1, B7H3, B7H4, TGF-β negativity membrane molecule and secretion TGF-β, IL-10 and PGE2 immunosuppression molecule.
10. purposes of repairing the mescenchymal stem cell of radioactivity enteric epithelium damage for orientation conveying Rspo1 according to claim 7, is characterized in that: people Rspo1 protein molecule is promoting the application in intestinal crypts stem cells hyperplasia and differentiation.
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| CN113215097A (en) * | 2021-06-24 | 2021-08-06 | 成都中医药大学 | High-proportion B7H4 positive umbilical cord mesenchymal stem cells, and culture method and culture solution thereof |
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