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1、诱导骨髓间充质干细胞向心肌样细胞分化实验探究作者:郭茂娟姜希娟马东明范英昌【摘要】目的观察乳鼠心肌细胞条件培养液、SalB、5氮胞昔(5aza)及SalB联合5aza体外诱导骨髓间充质干细胞(MSCs)分化为心肌样细胞的作用及其机制。方法分离培养及鉴定大鼠MSCs,培养乳鼠心肌细胞,收集条件培养液对其进行分组诱导:①SalB(终浓度为250Ug/L),②5aza组(终浓度为10ug/L),③SalB联合5aza组(终浓度分别为250ug/L与10umol/L),④乳鼠心肌细胞条件培养液,⑤未加诱导剂的阴性对照组。选取MSCs进行诱导,诱导周期为4wo倒置相差显微镜下观察细胞的生
2、长情况及形态变化,免疫细胞化学法鉴定心肌特异性肌钙蛋白T(cTnT)表达,实时荧光定量RTPCR法检测心肌早期基因NKX2.5、GATA4mRNA的相对表达量。结果细胞形态学显示,诱导后的MSCs体积增大,增殖减慢,并出现肌管样结构;免疫细胞化学结果显示,诱导后的MSCs表达心肌特异性蛋白cTnT,与5aza组相比,SalB组和条件培养液两组蛋白表达较低,SalB联合5aza组蛋白表达升高;实时荧光定量RTPCR结果显示除阴性对照组外,其他四组表达心肌分化早期基因,SalB联合5aza组诱导后的MSCs的心肌早期分化基因表达明显高于其他三组。结论SalB联合5aza诱导MSCs
3、表达心肌特异性肌钙蛋白cTnT;上调心肌早期基因NKX2.5、GATA4mRNA的表达,证实其能够向心肌细胞分化能力;条件培养液不能上调心肌细胞的早期基因和蛋白,不能诱导其分化。【关键词】骨髓间充质干细胞;诱导分化;丹酚酸B;5氮胞昔[Abstract】ObjectiveToexploretheroleandmechanismofbonemarrowmesenchymalstemcells(MSCs)differentiatedintomyocardialcellsinducedbyneonatairatmyocardialcellsconditionedmedium,salv
4、ianolicacidB(SalB),andSalBcombinedwith5azacytidine・MethodsMSCsofratswereseparated,culturedandindentified.Neonatairatmyocardialcellswereculturedandconditionculturefluidwascollected.MSCswereinducedbySalB(terminalconcentration250ug/L),5azacytidine(5aza)(250Pg/L),SalBcombinedwith5aza(250ug/Land1
5、0Pmol/L),Neonatalratmyocardialcellsconditionculturefluid,andwihoutanyinducerrespectivelyfor4weeks・Cellsgrowthconditionandmorphologicchangewereobservedbyinvertedphasecontrastmicroscope,cTnTexpressionwasindentifiedbyimmnocytohistochemicalassay,andmyocardialearlygeneNKX2.5,andGATA4mRNAexpressio
6、nweredetectedbyRTPCR・Results①Cellmorphologicobservation:afterinducing,MSCsvolumeenlarged,proliferationspeedsloweddownandappearredstruetureofmyotube.©ExpressionofcTnT:cTnTwasdetectedininducedMSCs.Comparedwith5azagroup,thecTnTexpressionsinSalBgroupandconditionculturefluidgroupwerelowinSalBgrou
7、pandconditionculturefluidgroupandhighinSalBcombinedwith5azagroup・©Expressionsofmyocardialearlygene:myocardialearlygenewasmeasuredinother4groupsexceptnegativecontrolgroup,andwashighinSalBcombinedwith5azagroupthaninother3groups・ConelusionsSalBcombine