The Study Of Influential Factors Of Proliferation, Secretion, Apoptosis, Migration And Adhension Of Human Marrow Mesenchymal Stem Cell And Their Mechanisms

Bone marrow-derived cells with the self-renewal ability and multipotency ofdifferentiation have shown the capacity of regenerating damaged myocadial tissue.Bone marrow mesenchymal stem cells (MSCs) are a population of multipotent Adultstem cells resided in bone marrow besides hematopoietic stem cells (HSCs),and theyare likely to be an ideal source for transplantation to repair damaged myocardium.The studies of MSCs to treat cardiac dysfunction resulted from ischemic heart diseaseand cardiac dysfunction are attracking more and more focus,but they are still in thephase of beginning.We studied the effects of different drugs on proliferation,secretion,apoptosis,migration and adhension of human marrow mesenchymal stemcells,and explored the correlative cell signaling pathways.Partâ… Isolation,culture,identification and induction of human marrowmesechymal stem cellsObjective:The study is to establish the isolation,culture,purification,proliferationmethod of human marrow mesechymal stem cells (MSCs) in vitro and examine theirphenotype.We induced MSCs to cardiomyocytes-like cells by the use of5-azacytidine (5-aza) and identified them.Methods:MSCs were isolated from healthy volunteers' marrow according to densitygradient centrifugation and purified by their characteristic of adhering to plasticsurfaces.The MSCs in culture were observed under phase microscopy and examinedthe expression of surface markers CD34,CD105 and CD106.After 6 passages,MSCswere treated by 5-aza,observed under phase microscopy and assessed formorphological change.The differentiation of MSCs was evaluated byimmunochemical staining for myosin heavy chainsβand cardiac troponin T (cTnT).Results:We obtained many MSCs through the combination of density gradientcentrifugation and adherence method.After passage,the cells were further purified.2×10⁵cells were seeded in 25 cm² cell culture flask with standard culture medium of10% fetal calf serum,and were passaged after 80%～90% confluence.High purity and active proliferation MSCs can maintain their biological characters withoutdifferentiation.After two weeks of primary culture,cells were passaged for the firsttime.Then they were passaged every 7 days and displayed a rather homogenouspopulation of fibroblast-like cells.No significant morphological changes wereobserved after 6 passages.After two weeks treatment of 5-aza to inducedifferentiation,cells at passage-6 enlarged and connected tightly withcardiomyocyte-like morphologies.Myosin heavy chainsβand cardiac troponin Twere negative before induction,and about 21% of cells became positive afterinduction.Conclusion:We could purify human MSCs in vitro by the combination of densitygradient centrifugation and adherence method.After two weeks induction of 5-aza,MSCs differentiated to cardiomyocyte-like cells at morphology and proteinexpression level.Partâ…¡:Effects of rhEPO,pravastatin,rhodioside and antiplatelet drugs onproliferation,secretion and differentiation of human mesenchymal stem cellsObjective:To investigate the effects of rhEPO,pravastatin,rhodioside andantiplatelet drugs on proliferation,secretion of VEGF and differentiation of humanmesenchymal stem cells.Methods:After being cultured in vitro,human MSCs were treated with drugsdescribed above.Then cellular growth curve was plotted.After treated by drugs,MSCs were observed with phase contrast microscope to find morphological changes.Cell proliferation was assessed by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay,level of VEGF of culturemedium was detected by enzyme-linked immunosorbent assay (ELISA),and CD34,CD105,CD 106 surface antigens of hMSCs were analyzed by flow cytometry.Afterpretreated by drugs,MSCs were induced by 5-aza and the differentiation efficiencywas detected by immunochemistry staining for anti-cardiac troponin T monoclonalantibody.Results:(1) Cell proliferation:The stage of latency of passage cells was about 48h.Day 4 to day 6 after passage,MSCs entered the exponential phase of growth,and then went into plateau phase at day 7.No significant changes of morphology and surfaceantigens were observed in human MSCs after treated by drugs.OD₅₇₀ values ofhMSCs treated by rhEPO (0.01U/mL～10 U/mL),pravastatin (0.2μmol/L～10μmol/L),rhodioside (0.2 mg/L～2 mg/L),clopidogrel (0.02μmol/L～40μmol/L) or ticlopidine(0.02μmol/L～40μmol/L) were higher than control group (P＜0.05),while OD₅₇₀ valuesof aspirin group (60μmol/L～2000μmol/L) were lower than control group (P＜0.05).(2) VEGF level:Treated by high dose rhodioside (5mg/L),clopidogrel (40μmol/L)orticlopidine (40μmol/L),VEGF level from hMSCs was lower than that of controlgroup (P＜0.01),but VEGF level of rhEPO,high dose pravastatin (100μmol/L ),lowdose rhodioside (0.2mg/L) and ticlopidine (0.02μmol/L) group was higher thancontrol group (P＜0.05),and there was no significantly difference of VEGF levelamong aspirin group,low dose pravastatin (0.2μmol/L) ,clopidogrel group(0.02μmol/L) and control group.(3) Cell differentiation:Six drugs had no obvious effect on cell surface antigens(CD34,CD105,CD106) expressed by hMSCs.After pretreated by six drugs andinduced by 5-aza,there was no significantly difference of cTnT positive rate of humanMSCs among control group and drug groups (P value was 0.597,0.952,0.325,0.141,0.830 and 0.072 respectively).Conclusion:RhEPO,pravastatin,rhodioside,clopidogrel and ticlopidine improveproliferation of human MSCs,but aspirin inhibits it.High dose rhodioside,clopidogrel and ticlopidine suppress VEGF secretion of human MSCs,while rhEPO,high dose pravastatin,low dose rhodioside and ticlopidine promote it.These drugshave no obvious effect on differentiation of human MSCs.Partâ…¢:Effects and mechanisms of rhEPO and pravastatin on proliferation,secretion,apoptosis,migration and adhesion of human mesenchymal stem cellsObjective:To investigate the effects and correlated cell signaling pathways of rhEPOand pravastatin on proliferation,secretion,apoptosis,migration and adhesion ofhuman mesenchymal stem cells in vitro.Methods:After being cultured in vitro,human MSCs were treated with rhEPO andpravastatin,phosphorylation of ERK 1/2,p38 MAPK and PI3K/Akt were detected by Westem blot.After pretreatment with inhibitor or agonist of ERK 1/2,p38 MAPK andPI3K/Akt,cell proliferation was assessed by MTT colorimetric assay,level of VEGFof culture medium was detected by ELISA,cell apoptosis was determined by flowcytometry,migration assay was performed in Transwell chambers,adhesion assay wasperformed by plastic dishes.Results:RhEPO and pravastatin could increase phosphorylation of PI3K/Aktpathway of human MSCs,but reduce phosphorylation ofp38MAPK.And they haveno obvious effect on ERK1/2 pathway and total proteins of Akt and p38MAPK.Theimprovement in cell proliferation was partly reverted in rhEPO group (P＜0.05) andwas abolished in pravastatin group when MSCs were pretreated with the p38MAPKagonist anisomysin,whereas no effects were observed using PI3K/AKT inhibitorLy294002.RhEPO's effect on VEGF secretion was weaken by precondition ofLy294002 or anisomycin (P＜0.01),and pravastatin's effect was partly reversed byLy294002 (P＜0.01) rather than anisomycin.RhEPO and pravastatin could decreasehuman MSCs apoptosis induced by H₂O₂ (P＜0.01) and the inhibitory effect wasabrogated by Ly294002 but not anisomysin.The number of cells crossing the filterwas significantly increased in rhEPO and pravastatin group (P＜0.01).This effect wascompletely inhibited by concomitant incubation with Ly294002.After treated withrhEPO and pravastatin,adherent cells increased significantly (P＜0.01).NeitherLy294002 nor anisomycin could inhibit this effect.Conclusion:RhEPO and pravastatin could increase the proliferative,VEGF secretion,migratory and adhesive capacities of human MSCs;meanwhile,they could protectMSCs from apoptosis.The inhibition ofp38MAPK pathway was related with twodrugs effects on proliferation and rhEPO effect on VEGF secretion.Activation ofPI3K/Akt pathway was involved in the effects of rhEPO and pravastatin on VEGFsecretion,migration and apoptosis.The effects of two drugs on adhesion capacityhave nothing to do with PI3K/Akt and p38MAPK pathway.