CN102634500A - Genetic engineering application of quick decidua and placenta mesenchymal stem cells (MSC) obtaining and micro-molecular modifying - Google Patents
Genetic engineering application of quick decidua and placenta mesenchymal stem cells (MSC) obtaining and micro-molecular modifying Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicine, and discloses a quick and effective method for obtaining decidua and placenta mesenchymal stem cells (MSC) and modifying the MSC with micro-molecules such as micro ribonucleic acid (RNA) and progesterone hormone in vitro. The method is characterized in that by adopting an optimized decidua, placenta tissue digestive enzyme formula and a digestive cells wall-sticking way, the decidua and placenta of the newborn fetus can be used as the resources to obtain the decidua and placenta MSC quickly and effectively and the MSC can be modified with miRNA143, miRNA181a and progesterone, thus the immunomodulatory capacity of the MSC can be further strengthened. The existing method for obtaining the decidua and placenta mesenchymal stem cells is optimized, and the MSC is modified with RNAs and progesterone hormone for the first time, thereby effectively improving the immunomodulatory capacity of the MSC. The invention provides a scientific, quick and effective obtaining and modifying method for the scientific research and clinical application of the decidua and placenta MSC.
Description
Technical field:
The present invention is the method that a kind of decidua fast and effectively, placenta MSC obtain and modify; Specifically utilize cell culture technology and rational tissue management technique to improve decidua, placenta MSC yield and the speed of obtaining; Rapid identification; And utilize small molecules such as micro ribonucleic acid and progesterone that MSC is modified, further improve the method for MSC immunoloregulation function.
Technical background
Mescenchymal stem cell is to belong to mesoblastic one type of multipotential stem cell, mainly is present between reticular tissue and organ in the matter.MSC has powerful multiplication capacity and multidirectional differentiation potential, in suitable body or can be divided into various kinds of cell such as hematopoietic cell, myocyte, liver cell, scleroblast, chondrocyte, stroma cell under the external environment.MSC also has immunoloregulation function except the general biology characteristics with stem cell simultaneously.It is convenient relatively that this cell also has the source in addition, is easy to separation, cultivation, amplification and purifying, still has the stem cell characteristic after the amplification of repeatedly going down to posterity, and do not have the characteristic of immunological rejection.Therefore MSC has become the focus in the stem-cell research in recent years, has application promise in clinical practice in fields such as Immunological diseases treatment, HSCT, organizational project, genetically engineereds.
MSC can suppress lymphocytic propagation, has very strong immunoloregulation function.Experiment confirm in the body, MSC are transplanted survival time that can obviously prolong cutify and are reduced recessive allele HSCT GVHD, we also successful use MSC transplantation treatment tens of routine Patients with SLE.Think MSC at present through secreting various immune modulatory molecules, thereby form and suppress DC cellular immunization activity inducing immune tolerance in the body with the induced chimaera that contacts of immunocyte.
Present research shows that MSC can also treat ischemic disorders.MSC and peripheral hematopoietic stem cells combined transplantation can promote the hematopoiesis of patient with breast cancer and high-risk patients with acute myeloid leukemia to recover, and not relevant negative interaction has shown the ability that good short hematopoiesis recovers.Going back to the nest and implant, can shorten that to transplant the time that the back hemopoietic function of bone marrow recovers relevant after thereby this result of treatment and the MSC of MSC cell secrete various Hemopoietic factors and generate amplification in vitro that bone marrow matrix helps hemopoietic stem cell, HSCT.Find all in the research of rat brain obstruction of artery and myocardial infarction model that simultaneously the MSC cell has the effect that improves illness.
Simultaneously MSC also has the multidirectional differentiation potential of stem cell as a kind of stem cell, and MSC can treat that congenital bone forming is bad, metachromatic leukodystrophy (MLD) and hurley syndrome; In parkinsonism; Senile dementia, spinal cord injury, the treatment of burn also has report.
These results have pointed out MSC to have the wide range of therapeutic function, and present countries in the world are all dropped into a large amount of manpower, material resources and financial resources and carried out extensive research.But above-mentioned regimen all needs a large amount of MSC to transplant, and therefore sets up stable MSC resources bank fast and effectively and has very important meaning.
It is generally acknowledged that the content of MSC in myeloid tissue is the abundantest; But along with wearing out of age; The MSC number significantly reduces in the ABM, the proliferation and differentiation ability fails significantly; And having the potentially dangerous of virus infection, the collection of donor MSC difficulty makes that the research of seeking other alternative MSC " resources bank " beyond the marrow is brought into schedule simultaneously.At present, isolate MSC the tissue beyond marrow such as fat, embryo, amniotic fluid, placenta, decidua and umbilical cord.And in these tissues, decidua and placenta belong to Biohazard Waste, obtain the most easily, and to its research and use and do not relate to ethnics Problem, and have and stablize a large amount of sources, therefore have the value of research.Present research shows; Decidua and placenta mesenchyma stem cell are present in decidua and placenta jelly of Wharton and the blood vessel surrounding tissue; Cultivate the inoblast appearance of obtaining at this position; Express alpha-smooth muscle actin and multiple MSC affinity tag (CD29, CD44, CD51, CD105, SH2, SH3), but do not express endothelium or the special antigens c D34 of white corpuscle, CD45.Simultaneously in decidua and placenta mesenchyma stem cell substratum, add U-18496; MSC breaks up to the myocardial cell; Changing culture condition also can point out decidua and placenta MSC cell to have identical phenotype and differentiation potential with marrow MSC cell to adipocyte and osteocyte differentiation.
The method that is used for the separating mesenchymal stem cell at present mainly contains 4 kinds, i.e. fluidic cell separating method, magnetic bead partition method, density gradient centrifugation and adherent sieve method.Convection type cell sorting method and magnetic bead partition method need not select for use a plurality of surface markers to detect because of finding the surface marker that the mescenchymal stem cell specificity is very strong, investigator so far as yet; In addition, these two kinds of active influences of method pair cell are big, requirement for experiment condition is higher, and are unfavorable for the separation sorting of cell, so not widespread use as yet at present.Density gradient centrifugation is owing to need accurate instrument and suitable parting liquid, and pair cell also has certain influence, and is not suitable for large-scale production application.And adherent sieve method, the cellular form homogeneous that is obtained, rate of propagation is fast, and growth conditions is stable, and easy and simple to handle, and cost is low, is expected to become practical working method.Pertinent literature has been reported the acquisition methods of decidua and placenta MSC cell, and it is relatively low to obtain efficient, and speed is slower.We have improved the traditional endotheliocyte digestion method and the prescription of digestive ferment, adopt and partly organize adherent method can obtain decidua and placenta MSC cell in a large number fast.And very high acquisition efficient and yield arranged.Present method is simple, more helps being used for production practice.
MSC can be used as the good carrier that carries foreign gene through genetic modification in addition, the people has been arranged cytokine, imports in the MSC cell like IL23, EPO etc., gets into acquisition long-time stably express in back in the body.Someone imports the gene of IFN β in the MSC, finds that the microenvironment of tumour makes MSC implant easily, and the MSC that gets in the tumour suppresses tumor growth through secretion IFN β cytokine, has shown that MSC has the tumor biotherapy function of possibility.Pointed out through rational genetic modification mode, can further improve the function of MSC, more reasonably be used for practical application.
The present invention utilizes micro ribonucleic acid and progesterone that MSC is modified on decidua that obtains and placenta MSC basis, and the immunoloregulation function of MSC is improved, and is more conducive to clinical application.
Summary of the invention:
The purpose of this invention is to provide a kind of through optimizing decidua and placenta tissue digestive ferment prescription and the adherent mode of peptic cell; Effectively utilize Biohazard Wastes such as decidua and placenta to be resource acquisition decidua and placenta MSC and MSC evaluation fast fast; And the MSC cell is reasonably modified the method for improving the MSC immunoloregulation function through small molecules such as micro ribonucleic acid and hormones, specific as follows:
Clip cuts open the palace and produces healthy puerpera's decidua and placenta, places the PBS of aseptic precooling to preserve.Decidua and placenta are confirmed to use after the safety through strict pathogen detection.
In the Bechtop, decidua and placenta tissue be put in the DMEM/F12 substratum be cut into 2mm
3About tissue block.
The enzyme that conventional decidua and placenta tissue digestion are used is the mixed solution of collagenase or collagenase and pancreatin, influences the primary cell state if want with organizing complete digestion often to need the long time.And if digestion time is shorter, can not then can influence separation efficiency with organizing complete digestion.And than higher, organizing that bulk does not shred usually can not complete digestion to the pulverizing of tissue block or the requirement that shreds for this digestion method.Simultaneously, use collagenase or collagenase to become thick with the tissue of the mixed solution digestion of pancreatin more, need a large amount of damping fluid dilutions, repeatedly flushing just can be obtained corresponding cell, has more strengthened the difficulty that the MSC cell obtains.Therefore; We have disposed a kind of enzyme that is specifically designed to decidua and placenta tissue digestion: CDH digestive ferment, this enzyme can digest decidua and placenta tissue in the short period of time fully, and be lower to the requirement that tissue block is pulverized; And Digestive system is thickness not, more easily the centrifugal MSC cell that obtains of flushing.
After conventional decidua and the placenta tissue digestion, can filter, obtain single cell suspension through 100 eye mesh screens; It is adherent to be inoculated in Tissue Culture Flask then, and this method MSC obtains comparatively fast, but the MSC cell that obtains is generally less; And because the intermediate treatment process is too many, cell state is affected.Also have bibliographical information to obtain MSC through the method for tissue block adherent, but this method is too high to the tissue block processing requirements, the most tissues piece can not be adjacent to cell bottle wall, and generally needs 3 weeks just can obtain the MSC cell.Observation through us is found; After tissue digests through digestive ferment; The MSC cell is general agglomerating or stick to not complete digestion tissue, and the MSC cell that major part digested out after Digestive system was filtered through 100 eye mesh screens can not pass through screen cloth because of agglomerating, and efficient is obtained in influence.We have invented following method to this: the postdigestive tissue juice of CDH digestive ferment need not through screen cloth; Most of individual cells or MSC cell mass all can be adherent; Even the completely little block organization of not digestion is arranged; Through behind the CDH digestive ferment, also become and very easily be adjacent to cell bottle wall, can obtain the MSC cell fast.This method has combined the digestion of decidua and placenta tissue to obtain unicellular and the two-fold advantage of organizing adherent method, has effectively improved yield and the speed of MSC.
Concrete operating process is following: tissue that shreds and CDH mixed enzyme solution add in the 50ml centrifuge tube by 1: 1 mixed, place 37 ℃ of shaking tables, and the 200rpm vibration is about digestion 3h.Postdigestive organize PBS washing three times after, be resuspended in the DMEM/F12 substratum that contains 12% foetal calf serum, the T-25 culturing bottle is gone in the shop.
Cultivate after 2 days, discard not adherent cell and tissue, add the fresh DMEM/F12 substratum that contains 10% foetal calf serum, change liquid every other day.When cell grows to 80% abundance (about 8 days), cell dissociation first three the generation ratio of cell in 1: 1 is inoculated in the new T-25 culturing bottle.Cell dissociation all went down to posterity by 1: 3 later on.Can obtain first-generation MSC, about 2 * 10 on the 8th day
6Individual cell, but and the corresponding phenotype of streaming detection MSC.
The MSC that obtains is inoculated in 24 orifice plates with 35000 cells/well, and pre-miRNA181a and pre-miRNA143 molecule use 2000 pairs of MSC cells of Lipofectamine to carry out transfection with 15nM concentration respectively.The antagonist (anti-miR-181a and anti-miR-143) of miR-181a and miR-143 molecule is got final product the expression of reticent corresponding miRNA molecule with as above same method transfection.It is synthetic that pre-miRNA molecule and anti-miRNA inhibitor are (AMBION) company.After the transfection 48 hours, utilize real-time quantitative PCR to detect the mRNA expression level that related immune is regulated cytokine IL6, VEGF, IDO, TGF-beta and COX-2.And utilize ELISA test kit method to detect IL6 and vegf protein expression level in the cell conditioned medium.
MSC is with 10
5The density of/well is inoculated in 12 orifice plates, adds 10ul different concentrations of progesterone (10 behind the bed board respectively
-7M, 10
-6M and 10
-5M), 24h and 48h Q-PCR and ELISA detect the expression level of PGE2 and IL-6.And (MSC: DC=1: 10) be incubated at altogether in RMPI 1640 perfect mediums and (contain 10% calf serum), adding CpG stimulates 24h, and streaming detects the expression of DC surface C D40, CD86 and MHC-II molecule to get an amount of cell and DC.
Description of drawings:
The MSC cellular form in Fig. 1, decidua and placenta source: the decidua MSC (A) and the placenta MSC (B) that adopt present method to obtain cultivate the cellular form after 3 days, and the attached cell that the hair tonic of tissue block place goes out is divergence form shuttle shape.The single attached cell of part is also arranged and begin the division increment.(C) first-generation decidua MSC (C) and the placenta MSC (D) that adopt present method to obtain cultivate the form after 8 days, and the cell abundance reaches 80%, becomes the shuttle type fibroblast-like cells of the relative homogeneous of form, the growth of whirlpool shape.
Fig. 2, the quick phenotype of the MSC cell streaming of obtaining detect: third generation decidua MSC (A) that will in T-25 plastic culture bottle, cultivate and placenta MSC (B) cell dissociation obtain single cell suspension; Adopt machine testing on the mono-clonal fluorescence antibody difference mark, carry out data analysis with CellQuest software: the positive phenotype CD105 of the criterion of MSC cell, CD73, CD90, CD44, CD29, HLA-ABC positive rate are not less than 95%; Negative CD34, CD14, CD11b, CD19, HLA-DR, CD31 positive rate are not higher than 2%.
Fig. 3, utilize miRNA143 and miRNA181a that MSC is modified: (A) to use pre-miRNA molecule and anti-miRNAinhibitor molecule respectively the MSC cell to be modified; MiRNA143 and miRNA181a molecule are expressed with low by the difference high expression level in MSC; And through the PCR expansion; The amplified production electrophoresis detection, U6 is confidential reference items.(B) high expression level miRNA181a in the MSC cell extracts cell RNA, and uses real-time quantitative PCR to detect the mRNA expression level of IL6, VEGF-A and IDO1.The collecting cell supernatant, and with IL6 and vegf protein expression level in the ELISA test kit method detection cell conditioned medium.(C) difference high expression level miRNA143 and reticent miRNA143 in the MSC cell extract cell RNA, and use real-time quantitative PCR to detect the mRNA expression level of TAK1, COX-2 and IDO1.
Fig. 4, progesterone are handled the MSC impact cell: (A) use different concentrations of progesterone to handle MSC, and detect the protein expression level of COX-2 and PGE2 with WESTBLOT and ELISA respectively.(B) use different concentrations of progesterone to handle MSC, and use real-time quantitative PCR and ELISA test kit method to detect mRNA and the protein expression level of IL6.(C) MSC and DC cultivate altogether, and adding CpG stimulates 24h, and streaming detects the expression of DC surface C D40, CD86 and MHC-II molecule, and carry out data analysis with CellQuest software.
Embodiment:
The objective of the invention is to utilize cell culture technology and rational tissue management technique to improve and obtain decidua and placenta MSC yield and speed, and Rapid identification, and through small molecules the MSC cell is reasonably modified and to be improved the MSC immunoloregulation function.The contriver has announced a kind of decidua and placenta tissue digestive ferment prescription and adherent mode of peptic cell and MSC evaluation fast of optimization, and the MSC cell is reasonably modified the method for improving the MSC immunoloregulation function through small molecules.With regard to specific embodiment concrete utilization of the present invention is described below.
Clip cuts open the palace and produces puerpera's decidua and placenta, places the PBS of aseptic precooling to preserve.
Decidua and placenta confirm that through strict pathogen detection (microbial pathogenes such as treponema pallidum, hiv virus (HIV), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, mycoplasma detect) use the back safely.Get rid of the decidua and the placental samples of factors such as premature labor, premature rupture of fetal membrane, chorioamnionitis, systemic lupus erythematous, ephritis, mellitus simultaneously, guarantee that the MSC that obtains is normal.
In the Bechtop, decidua and placenta taken out put into glass culture dish, clean, and decidua and placenta tissue be put in the DMEM/F12 substratum be cut into 2mm with surgical scissors with PBS
3About tissue block.
Configuration CDH digestive ferment (II Collagen Type VI enzyme 250U/ml, neutral protease 100U/ml, Unidasa 10U/ml), 37 ℃ are dissolved in the DMEM/F12 substratum, and the filter degerming of filtering 0.22 μ m is subsequent use.Digestive ferment is preferably joined existing usefulness at present, spends the shelf time above 1 week 4.
The tissue that shreds and CDH mixed enzyme solution by 1: 1 volume ratio mix with the 50ml centrifuge tube in; Place 37 ℃ of shaking tables; 200rpm vibration is about digestion 3h (treat tissue block digests basically get final product, the residual MSC that do not influence of the tissue block that minority is less obtains efficient).
With 4 ℃ in postdigestive tissue juice, the centrifugal 5min of 300g abandons supernatant, abandons supernatant, will precipitate with PBS to be resuspended in the 50ml fresh culture, and 4 ℃, the centrifugal 5min of 300g abandons clearly, and PBS washes twice, notes cell and tissue deposition not being discarded.Last abandons supernatant all over after centrifugal, deposition is resuspended in the DMEM/F12 substratum that contains 12% foetal calf serum, and the T-25 culturing bottle is gone in the shop.The shop can not use 100 eye mesh screens to filter before going into the Tissue Culture Flask culturing bottle, filters the acquisition efficient that can influence the MSC cell.(about 2cm
3The deposition of decidua and placenta digestion gained be laid in 1 T-25 culturing bottle)
Cultivate after 2 days, discard not adherent cell and tissue, add the fresh DMEM/F12 substratum that contains 10% foetal calf serum, change liquid every other day.The MSC cell can grow along adherent tissue block or adherent cell.When cell grows to 80% abundance (about 8 days), cell dissociation buffer (0.25% pancreatin+0.02% EDTA), in first three generation, be inoculated in the ratio of cell in 1: 1 in the new T-25 culturing bottle, to guarantee growth velocity.Cell dissociation all went down to posterity by 1: 3 later on.
Every 2cm
3Decidua and placenta, obtained first-generation MSC, about 2 * 10 on the 8th day
6Individual cell.
Morphological observation: primary cell and tissue block through present method obtains are how adherent in 2 days, cultivate the attached cell that promptly can be observed in 4-6 days to go out from the tissue block hair tonic, are divergence form shuttle shape.Also can be observed simultaneously the single attached cell of part and begin the division increment.After 1 time go down to posterity (8 days), the cell increment is very fast, becomes the shuttle type fibroblast-like cells of the relative homogeneous of form, the growth of whirlpool shape.Considerable change does not take place in 15 cellular fories of continuous passage.According to the report of document and other patents, obtaining first-generation MSC cell needed for 2 weeks, and success ratio is not high.Cell count that present method obtains and acquisition time and success ratio all obviously are superior to according to other documents and patent.
Streaming detects:
How the MSC that separates, increases identifies, is present question at issue.MSC can express kinds of surface antigen but be not special, but through checking that a plurality of surface markers can distinguish MSC and other cells, pertinent literature also has report.Flow cytometry is a kind of fast accurate detection means simultaneously, can confirm to obtain the phenotype of cell very fast and effectively.Avoid traditional passing through to detect MSC cytodifferentiation ability and identified long shortcoming consuming time.
The third generation MSC cell that in T-25 plastic culture bottle, cultivate obtains single cell suspension with cell dissociation buffer digestion, and cell counting.The MSC cell is resuspended to 1 * 10
7Cell count/milliliter, single cell suspension packing are gone in the streaming pipe, every pipe 100 μ l.The streaming fluorescence antibody that in each streaming pipe, adds respective markers, mixing.4 ℃ of lucifuges were hatched 30 minutes.Each streaming pipe adds the antibody of 500 μ l PBS flush awaies on unmarked, and 4 ℃, the centrifugal 10min of 300g abandons supernatant, abandons supernatant, triplicate.Subsequently with cell precipitation resuspended with 100 μ l PBS in.Flow cytometer detects corresponding phenotype, and through the corresponding software analysis.The positive phenotype CD105 of the criterion of MSC cell, CD73, CD90, CD44, CD29, HLA-ABC positive rate are not less than 95%; Negative CD34, CD14, CD11b, CD19, HLA-DR, CD31 positive rate are not higher than 2%
The MSC that aforesaid method is obtained is inoculated in 24 orifice plates with 35000 cells/well, and pre-miRNA181a and pre-miRNA143 molecule use 2000 pairs of MSC cells of Lipofectamine to carry out transfection with 15nM concentration respectively.The antagonist (anti-miR-181a and anti-miR-143) of miR-181a and miR-143 molecule is got final product the expression of reticent corresponding miRNA molecule with as above same method transfection.It is synthetic that pre-miRNA molecule and anti-miRNA inhibitor are (AMBION) company.
After the transfection 48 hours, draw cell conditioned medium and preserve subsequent use.Extract cell total rna with Trizol Reagent reagent.Becoming cDNA. with the total RNA of Revert AidTMFirst Strand cDNA Synthesis test kit counter-rotating 1ug utilizes real-time quantitative PCR to detect the mRNA expression level of relevant cell factor IL6, VEGF, IDO, TGF-beta and COX-2.And utilize ELISA test kit method to detect IL6 and vegf protein expression level in the cell conditioned medium.
The result shows, the mRNA of the IL6 of MSC, VEGF-A and IDO and protein expression level rise behind the high expression level miRNA181a in the MSC cell, and low expression miRNA181a does not influence the expression level of IL6, VEGF-A and IDO in the MSC cell.The mRNA of the TAK1 of MSC, COX-2 and IDO1 and protein expression level downward modulation behind the high expression level miRNA143 in the MSC cell, and TAK1, COX-2 and the IDO expression level in the MSC cell raises behind the low miRNA143 of expression.
MSC is with 10
5The density of/well is inoculated in 12 orifice plates, adds 10ul different concentrations of progesterone (10 behind the bed board respectively
-7M, 10
-6M and 10
-5M), 24h and 48h Q-PCR and ELISA detect the expression level of PGE2 and IL-6.And (MSC: DC=1: 10) be incubated at altogether in RMPI 1640 perfect mediums and (contain 10% calf serum), adding CpG stimulates 24h, and streaming detects the expression of DC surface C D40, CD86 and MHC-II molecule to get an amount of cell and DC.
The result shows: progesterone is handled detailed rising of ability of back MSC secretion PGE2 and IL-6; And after MSC that P4 handles and DC train altogether; All than MSC+DC all significantly reductions of culture group altogether, this shows that prompting P4 can strengthen the restraining effect of MSC to DC in the expression of DC surface MHC-II, CD40 and CD86.
Claims (4)
1. enzyme that is specifically designed to the digestion of decidua and placenta tissue: CDH digestive ferment; This enzyme in the short period of time can be fully with decidua and placenta tissue digestion; Requirement to tissue block is pulverized is lower, and Digestive system not thickness, the centrifugal MSC cell that obtains of flushing more easily.The characteristic of this Digestive system is to contain II Collagen Type VI enzyme 250U/ml, neutral protease 100U/ml, and Unidasa 10U/ml, 37 degree are dissolved in the DMEM/F12 substratum, and the filter degerming of filtering 0.22 μ m is subsequent use.Digestive ferment is preferably joined existing usefulness at present, spends the shelf time above 1 week 4.
2. method that obtains fast effectively decidua and placenta mesenchyma stem cell; It is characterized by utilization claim 1 described special-purpose decidua and placenta tissue digestive ferment prescription, decidua and the placenta that effectively utilizes newborn fetus fast is resource acquisition decidua and placenta MSC and the method identified of MSC fast.
3. the MSC modifying method of miRNA181a and miRNA143 is characterized by the modification of on decidua described in the claim 2 and placenta MSC, carrying out miRNA as required, changes the secretion of the MSC associated adjustment factor, and the immunoregulation capability of MSC is provided.
4. a progesterone is to the MSC modifying method, it is characterized by at the decidua described in the claim 2 and placenta MSC through after the hatching of progesterone, and MSC is further strengthened to the inhibit feature of DC immunocyte.
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| CN109423473A (en) * | 2017-08-21 | 2019-03-05 | 青岛瑞思德生物科技有限公司 | It is used to prepare the kit of placenta stem-cell |
| WO2022217878A1 (en) * | 2021-04-13 | 2022-10-20 | 广东唯泰生物科技有限公司 | Preparation method and recovery method for pariduval mesenchymal stem cells |
| WO2023044902A1 (en) * | 2021-09-27 | 2023-03-30 | 金涌长生医学生物科技股份有限公司 | Decidual placental mesenchymal stem cell and use thereof in preparation of pharmaceutical composition for promoting angiogenesis |
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| CN104711308A (en) * | 2015-04-09 | 2015-06-17 | 北京德明特生物技术有限公司 | Micromolecule substance for stimulating mesenchymal stem cells to secrete fibronectin |
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Application publication date: 20120815 |