鹿茸多肽诱导心肌干细胞向心肌细胞分化的机制研究

Study on the Mechanism of Differentiation from Cardiac Stem Cells into Cardiac Cells Induced by Cornu Corvi Polypeptides

目的:探讨鹿茸多肽诱导心肌干细胞(CSCs)向心肌细胞分化的机制。方法:从SD大鼠体内分离CSCs,鉴定后培养传代至P3代后,分为空白对照组、5-氮胞苷诱导组(3μmol/L)、鹿茸多肽诱导组(800μg/ml)及其联合诱导组,除空白对照组细胞只加含3%胎牛血清的培养液外,其余各组细胞加入含相应诱导剂的培养液,培养14 d。采用免疫组化法检测细胞表面心肌肌钙蛋白T(cTnT)表达,免疫印迹技术检测细胞cTnT、活化复制因子2(ATF-2)和肌细胞增强因子2C(MEF-2C)表达,实时定量聚合酶链式反应技术检测细胞cTnT和转录因子Nkx2.5、GATA4 m RNA表达。结果:成功制得CSCs,纯度>97%。与空白对照组(cTnT呈阴性)比较,各诱导组细胞cTnT呈阳性,cTnT、ATF-2、MEF-2C蛋白表达和cTnT、Nkx2.5、GATA4 m RNA表达均增强(P<0.05)。与5-氮胞苷诱导组比较,鹿茸多肽诱导组细胞cTnT、MEF-2C蛋白表达和cTnT、GATA4 m RNA表达均增强(P<0.05),联合诱导组细胞cTnT、ATF-2、MEF-2C蛋白表达和cTnT、GATA4 m RNA表达均增强(P<0.05)。结论:鹿茸多肽可能通过增强Nkx2.5、GATA4、ATF-2、MEF-2C等转录因子的表达来诱导CSCs向心肌细胞分化。

OBJECTIVE：To explore the mechanism of differentiation from cardiac stem cells（CSCs） into cardiac cells induced by Cornu Corvi polypeptides.METHODS：CSCs were isolated from SD rats,and cultured to P3 generation after identification and then divided into blank control group,5-azacitidine induction group（3 μ mol/L）,Cornu Corvi polypeptides induction group（800 μg/ml）and combination induction group.Except blank control group was cultured with culture solution containing 3%FBS,other groups were cultured with culture solution containing relevant derivant for 14 d.The expression of c Tn T was determined by immunohistochemical method;the expression of c Tn T,ATF-2 and MEF-2C were determined by immunoblot assay;m RNA expression of c Tn T,Nkx2.5 and GATA4 were detected by RT-PCR.RESULTS：CSCs were prepared successfully with purity〉97%.Compared with blank control group（c Tn T being negative）,c Tn T of induction groups were positive,and the protein expression of c Tn T,ATF-2 and MEF-2C and m RNA expression of c Tn T,Nkx2.5 and GATA4 were all increased（P〈0.05）.Compared with 5-azacitidine induction group,the protein expression of c Tn T and MEF-2C and m RNA expression of c Tn T and GATA4 were decreased in Cornu Corvi polypeptide groups（P〈0.05）;the protein expression of c Tn T,ATF-2 and MEF-2C and m RNA expression of c Tn T and GATA4 were all increased in combination induction group（P〈0.05）.CONCLUSIONS：Cornu Corvi polypeptides can induce differentiation of CSCs into cardiac cells by promoting the expression of Nkx2.5,GATA4,ATF-2,MEF-2C and other transcription factors.