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《誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向心肌樣細(xì)胞分化實(shí)驗(yàn)探究》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在工程資料-天天文庫。
1、誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向心肌樣細(xì)胞分化實(shí)驗(yàn)探究作者：郭茂娟姜希娟馬東明范英昌【摘要】目的觀察乳鼠心肌細(xì)胞條件培養(yǎng)液、SalB、5氮胞昔(5aza)及SalB聯(lián)合5aza體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(MSCs)分化為心肌樣細(xì)胞的作用及其機(jī)制。方法分離培養(yǎng)及鑒定大鼠MSCs,培養(yǎng)乳鼠心肌細(xì)胞，收集條件培養(yǎng)液對其進(jìn)行分組誘導(dǎo)：①SalB(終濃度為250Ug/L),②5aza組(終濃度為10ug/L),③SalB聯(lián)合5aza組(終濃度分別為250ug/L與10umol/L),④乳鼠心肌細(xì)胞條件培養(yǎng)液，⑤未加誘導(dǎo)劑的陰性對照組。選取MSCs進(jìn)行誘導(dǎo)，誘導(dǎo)周期為4wo倒置相差顯微鏡下觀察細(xì)胞的生
2、長情況及形態(tài)變化，免疫細(xì)胞化學(xué)法鑒定心肌特異性肌鈣蛋白T(cTnT)表達(dá)，實(shí)時(shí)熒光定量RTPCR法檢測心肌早期基因NKX2.5、GATA4mRNA的相對表達(dá)量。結(jié)果細(xì)胞形態(tài)學(xué)顯示，誘導(dǎo)后的MSCs體積增大，增殖減慢，并出現(xiàn)肌管樣結(jié)構(gòu)；免疫細(xì)胞化學(xué)結(jié)果顯示，誘導(dǎo)后的MSCs表達(dá)心肌特異性蛋白cTnT,與5aza組相比，SalB組和條件培養(yǎng)液兩組蛋白表達(dá)較低，SalB聯(lián)合5aza組蛋白表達(dá)升高；實(shí)時(shí)熒光定量RTPCR結(jié)果顯示除陰性對照組外，其他四組表達(dá)心肌分化早期基因，SalB聯(lián)合5aza組誘導(dǎo)后的MSCs的心肌早期分化基因表達(dá)明顯高于其他三組。結(jié)論SalB聯(lián)合5aza誘導(dǎo)MSCs
3、表達(dá)心肌特異性肌鈣蛋白cTnT；上調(diào)心肌早期基因NKX2.5、GATA4mRNA的表達(dá)，證實(shí)其能夠向心肌細(xì)胞分化能力；條件培養(yǎng)液不能上調(diào)心肌細(xì)胞的早期基因和蛋白，不能誘導(dǎo)其分化?！娟P(guān)鍵詞】骨髓間充質(zhì)干細(xì)胞；誘導(dǎo)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
