鹿茸多肽对心肌干细胞凋亡和线粒体膜稳定性的影响

Effect of pilose antler polypeptides on apoptosis and membrane stability of mitochondria in cardiac stem cells

背景:心肌干细胞在体外经鹿茸多肽诱导后可以分化为心肌细胞这一研究成果为其治疗心肌损伤提供了新思路。目的:观察鹿茸多肽对心肌干细胞凋亡和线粒体膜稳定性的影响,探讨其作用机制。方法:选取出生2 d的雄性Wistar大鼠,提取心肌干细胞,以培养皿作为实验单位,共计分为以下4组(n=12):鹿茸多肽组:使用鹿茸多肽(质量浓度为800 mg/L)进行诱导;联合组:使用5-氮胞苷(浓度为3μmol/L)和鹿茸多肽(质量浓度为800 mg/L)联合诱导;5-氮胞苷组:使用5-氮胞苷(浓度为3μmol/L)进行诱导;空白对照组:加入等量缓冲液进行诱导。诱导48 h后流式细胞仪检测心肌干细胞的凋亡率,免疫荧光检测心肌干细胞的线粒体膜电位,Western blotting方法检测心肌干细胞Nkx 2.5、GATA4、ATF-2、MEF-2C的表达水平。结果与结论:(1)细胞培养2周后,出现大量的小、圆、亮细胞,心肌干细胞集落形成;(2)与空白对照组比较,5-氮胞苷组心肌干细胞凋亡率明显升高(P<0.05),线粒体膜电位明显降低(P<0.05);与5-氮胞苷组比较,鹿茸多肽组、联合组心肌干细胞凋亡率明显降低(P<0.05,P<0.01),线粒体膜电位明显升高(P<0.05,P<0.01);(3)与5-氮胞苷组比较,鹿茸多肽组及联合组Nkx2.5的表达水平明显升高(P<0.05),各组GATA4、ATF-2表达水平不高,且差异无显著性意义(P>0.05),MEF-2C表达在中等水平,各组间差异无显著性意义(P>0.05);(4)结果表明,鹿茸多肽可降低心肌干细胞凋亡率,提高线粒体膜稳定性,其作用机制在一定程度上与提高Nkx 2.5表达有关,是否与GATA4、ATF-2、MEF-2C有关尚待进一步研究。

BACKGROUND： Cardiac stem cells can differentiate into cardiomyocytes in vitro under induction of pilose antler polypeptides, which provides a new therapeutic idea for myocardial injury.OBJECTIVE：To explore the effect of pilose antler polypeptides on the apoptosis rate and membrane stability of mitochondria in cardiac stem cells.METHODS：We chose healthy male Wistar rats born 2 days to extract cardiac stem cells.The culture dish was used as the experimental unit,and extracted cells were divided into the following four groups（n=12）.Blank control group：The same amount of buffer was added for induction;5-azacytidine group：induced with 5-azacytidine（3μmol/L）;pilose antler polypeptides group：induced with pilose antler polypeptides（800 mg/L）;combined group：induced with pilose antler polypeptides（800 mg/L）and 5-azacytidine（3μmol/L）.After 48 hours induction,apoptosis rate of cardiac stem cells in each group was detected with flow cytometry.The membrane potential of mitochondria in cardiac stem cells was detected with immunofluorescence.The expression level of Nkx 2.5,GATA4,ATF-2,and MEF-2C in cardiac stem cells was detected using western blot assay.RESULTS AND CONCLUSION：There were small,round and bright cells after 2 weeks culture,and cell colonies of cardiac stem cells formed.The apoptosis rate of cardiac stem cells in the 5-azacytidine group increased significantly（P〈0.05）and its membrane potential decreased significantly compared with the blank control group（P〈0.05）.The apoptosis rate of cardiac stem cells in the pilose antler polypeptides group and combined group decreased significantly compared with the 5-azacytidine group（P〈0.05,P〈0.01）,and their membrane potential increased significantly（P〈0.05,P〈0.01）.The results of western blot showed that the expression level of Nkx2.5 increased significantly in the pilose antler polypeptides group and combined group compared with the 5-azacytidine group（P〈0.05）,while the expression levels of GATA4 and ATF-2 in each group were low and there were no significant differences among groups（P〈0.05）.The expression level of MEF-2C in each group was at a middle level,and there were no significant differences among groups（P〈0.05）.To conclude,our experimental findings indicate that pilose antler polypeptides could decrease the apoptosis rate and improve membrane stability of mitochondria in cardiac stem cells.The mechanism may be related to the increased expression of Nkx 2.5,but whether the mechanism is related to GATA4,ATF-2 and MEF-2C needs to be further studied.