Isolation And Culture Of Human Bone Marrow Mesenchymal Stem Cells And Induced Differentiation Of VEGF

Objective: Cerebral ischemia is a frequent cerebrovasulardisease,it has high morbidity and mortality. Beacuse the bloodcirculation is obstructed by various factors, the central nervous systemis destroyed. The patho-physiological changes of injured region oflocal cerebral vessels play a important role in the reparation,anagenesis of the central nervous system after cerebral ischemia. At present, the therapy in cerebral ischemia includes two aspects:one is the recovery of blood circulation around the region of ischemia,the other is the reparation, anagenesis, structure restoration andfunctional restoration of nerve tissues. To improve the ischemia andoxygen deficiency in the surrounding is the precondition of repairingthe nerve tissue. So, only by recovering the two hands that can cure orabate the destroy of cerebral ischemia. It will be an effective strategyto treat cerebral ischemia through implanting stem cells which willdifferentiate vascular tissues or will be used as genetic carrier of thosefactors which can promote angiogenesis. The resours of MSCs is extensive, the fractional cultivation iseasy. The ability of amplification of MSCs is high in vitro. MSCs candifferentiate to many non-haematogenesis tissue and can be easilytransfected by exogenous gene and express. It consider that MSCs aredeal target cells of cell therapy and gene therapy. The other importantrole in the therapy of cerebral ischemia is VEGF. The function ofpromoting the vasiformation of VEGF has already been cofirmed bymany studys. beacause the biologic half-life of VEGF is short, thetherapeutic effect is transient. Now, people study the gene transferthepray of VEGF. This study will connect the forte of MSCs andVEGF, isolate and culture human bone mesenchymal stem cells(MSCs) and detect their immunophenotypes and investigate theinduced differentiation of VEGF (vascular endothelial growth factor,VEGF) in vitro through VEGF165 transfecion, which can promoteAngiogenesis, so the experiment will contribute to cell transplantationand gene therapy in cerebral ischemia.Method: Primary culture of MSCs: fresh bone marrow obtainedfrom healthy donor volunteers were isolated and cultured by percollgradient centrifugation and adherence to plastic flask. The cellularmorphologhy and growth was observed under inverted microscopeeveryday. When the MSCs reached to approximately 80% confluence,they were passaged and proliferated by monoclonal culture.Immunophenotypes of MSCs were detected by flow cytometrytechniques. pcDNA3.0-VEGF165 plasmids were duplicated,amplificated and extracted, sublimated in prokaryotic cell of Bacilluscoli DH5α. MSCs were transfected by means of lipofectamine mediamethod. Then immunofluorescence staining was employed, useingprimary antibodies to VEGF and CD31 (1∶50) and Secondaryantibodies to VEGF and CD31 labeled respectively by FITC and cy3and observed under laser scanning confocal microscope. AfterVEGF165 transfection, MSCs took on endothelial differentiationbecause the expression of CD44 was drastically decreased and CD31increased.Results: After one week of primary culture of MSCs, thehematopoietic cells disappeared, the adherence cells increased andshowed on fusiform shape, stretched out gross cell process. After twoweeks, MSCs confluenced to monolayer. Fusiform ecphyma becamelonger. The arrangement of MSCs took on evidently directionality,such as vortex, reticulate, radiat. The immunophenotypes of MSCswere positive for CD44 (73.90%) CD29, but negative for CD34,CD31 (4.37%) CD45. After VEGF165 transfection, MSCs took onendothelial differentiation because the expression of CD44 (41.67%)was drastically decreased and CD31 (58.12%) increased. MSCs wereinduced and differentiated to endothelial cells. The conclusion ofimmunofluorescence staining was that the antibodies to VEGF labeledby FITC made MSCs show green and CD31 labeled by cy3 madeMSCs show red. It proved that MSCs after VEGF165 transfectiontook on endothelial differentiation and could express VEGF.Conclusion: MSCs were isolated and cultured by percollgradient centrifugation and adherence to plastic flask successfully inthis experiment. The classics method was confirmed. Theamplification time of primary MScs was 10~15 days. Theimmunophenotypes of MSCs were positive for CD44 CD29, butnegative for CD34,CD31,CD45 by flow cytometry. MSCs weretransfected by means of lipofectamine media method. After VEGF165transfection, MSCs took on endothelial differentiation because theexpression of CD44 was drastically decreased and CD31 increased. Itwas confirmed that MScs after transfection could express VEGF bythe method of immunofluorescence stain. It can direct the cell therapyand promoting vessels restoration of cerebral ischemia. The laterstage of animal experiment and clinical experiment can continue andevolve.