gata4基因对促进骨髓间充质干细胞旁分泌作用的分析 word格式.docxVIP

MSCs的存活情况。结果:1.成功分离纯化骨髓MSCs，细胞接种24h后开始贴壁，呈较大的椭圆形，48h后细胞贴壁逐渐增多，呈均匀一致的长梭状，方向较规律，呈平行或旋涡状生长；2.实时定量PCR、WesternBlot和免疫组化方法结果显示GATA-4的mRNA和蛋白质在MSCGATA-4组中呈过量表达。3.缺氧状态下细胞比常规氧下细胞分泌VEGF、HGF和IGF-1明显增多（P0.05）；常规氧下，MSCGATA-4组VEGF和IGF-1的表达量明显较MSCs和MSCnull组升高（P0.05），而HGF表达量未见明显差异（P0.05）；缺氧状态下，MSCGATA-4组的VEGF和IGF-1的表达量明显较MSCs和MSCnull组升高（P0.05），而HGF表达量未见明显差异（P0.05）。4.与加入抗体之前相比，分别加入VEGF和IGF-1中和抗体后，各组细胞的MTT摄取率均降低，膜联蛋白V阳性细胞率和LDH释放量均升高（P0.05）；缺氧状态下，加入中和抗体后，与MSCs和MSCnull组相比，MSCGATA-4MTT摄取率较高，膜联蛋白V阳性细胞率和LDH释放量均较低（P0.05）；缺氧MSCs与缺氧MSCnull组比较，存活情况差异无统计学意义（P0.05）。结论：1.通过基因转染技术可以成功将骨髓MSCs高效过量表达GATA-4。2.过表达GATA-4的骨髓MSCs可以促进其分泌VEGF、HGF和IGF-1。3.加入VEGF和IGF-1中和抗体后MSCs存活明显受抑制，而GATA-4过表达可以改善细胞存活，推测GATA-4通过干扰MSCs的旁分泌机制影响细胞的存活。4.GATA-4基因转染MSCs可通过影响旁分泌作用促进细胞存活，可为MSCs移植治疗缺血性心脏病提供新的方法。关键词：GATA-4；骨髓间充质干细胞；旁分泌作者：黄美娇指导老师：陈弹教授StudyonGATA-4geneinimprovingtheparacrineeffectofbonemarrowmesenchymalstemcellsAbstractStudyonGATA-4geneinimprovingtheparacrineeffectofbonemarrowmesenchymalstemcellsAbstractObjective:Toinvestigatetheeffectofover-expressedGATA-4ontheparacrineeffectofmesenchymalstemcells(MSCs).Methods:MSCswereisolatedandculturedbyadherentculturemethodandidentifiedbyflowcytometer(FCM).MSCsweretransfectedwithGATA-4(MSCGATA-4)byusingapMSCVretroviralexpressionsystem,andnullvectortransfection(MSCnull)weretakenasthecontrol.GATA-4mRNAandproteinofMSCswereanalyzedbyReal-timequantitativePolymeraseChainReaction(RT-PCR),Westernblottingandimmunehistochemistry.MSCsweredividedinto：normoxicgroups:MSCs,MSCnullandMSCGATA-4group;hypoxicgroups:MSCs,MSCnullandMSCGATA-4group.GrowthfactorssecretedinMSCsmedium,suchasvascularendothelialgrowthfactor(VEGF),hepatocytegrowthfactor(HGF)andinsulingrowthfactor-1(IGF-1)weredetectedbyELISA.Afterculturingfor48hours,thecellularsurvivalwasevaluatedbyrateofMTTcellproliferation,positiverateofAnnexinVandlactatedehydrogenase(LDH)release.AfteraddingVEGFandIGF-1neutralizationantibodiesfor48hours,thesurvivalofMSCswasevaluatedbythesamemethodagain.Results:MSCshavebeenseparatedandpurifiedsuccessfully.Sp