胚胎干细胞条件培养基对过氧化氢引起心肌细胞凋亡的影响

Embryonic stem cell conditional medium inhibits HO induced cardiomyocyte apoptosis

目的研究胚胎干细胞条件培养基是否可以抑制过氧化氢引起的心肌细胞凋亡及其潜在的作用机制。方法收集培养小鼠胚胎干细胞24 h的培养液作为胚胎干细胞条件培养液,将大鼠乳鼠原代心肌细胞随机分为四组:空白对照组、H2O2(100μmol/L)处理组、对照培养液组和胚胎干细胞条件培养液组,其中对照培养液组和胚胎干细胞条件培养液组分别在原代心肌细胞中加入胚胎干细胞培养基、胚胎干细胞条件培养基后,再用100μmol/L H2O2处理4 h。通过MTT方法检测心肌细胞活力;通过细胞活性/毒性试剂盒检测心肌细胞存活情况;通过TUNEL凋亡试剂盒和电镜技术检测心肌细胞凋亡情况;通过激光共聚焦显微镜检测细胞内钙离子变化;通过Western blot法检测凋亡相关蛋白Bcl-2和Caspase3的表达情况。结果 MTT结果显示,与H2O2组相比,ES-CM组心肌细胞活力下降的现象明显得到抑制(P<0.05)。细胞活性/毒性试剂盒检测结果表明,与H2O2组相比,ES-CM组活细胞的百分数下降的现象明显得到抑制(P<0.05)。TUNEL凋亡试剂盒检测结果表明,与H2O2组相比,ES-CM组细胞凋亡百分数增加的现象明显得到抑制(P<0.05)。共聚焦显微镜检测的数据表明,与H2O2组相比,ES-CM组[Ca2+]i强度增加的现象明显得到抑制(P<0.05)。Western blot结果表明,与H2O2组相比,ES-CM组Bcl-2蛋白表达明显增加(P<0.05),Caspase3蛋白表达明显减少(P<0.05),而胚胎干细胞培养基组与H2O2处理组相比无显著变化(P>0.05)。结论胚胎干细胞条件培养基通过影响凋亡相关蛋白Bcl-2、Caspase3抑制H2O2诱导的心肌细胞凋亡。

Objective To investigate if embryonic stem cells conditional medium(ES-CM) can inhibit neonatal rat cardiomyocytes apoptosis in response to oxidative stress and the underlying mechanism. Methods The rat embryonic stem cells were pre-cultured with embryonic stem cell culture medium(ES-M) for 24 hours. Then the culture medium was collected as ES-CM. NRCMs were randomly divided into four groups: blank control group, H2O2(100 μmol/L) treatment group, control culture solution group, and mouse embryonic stem cell conditional medium group. Cardiomyocytes were pre-treated with ES-CM or ES-M, then treated with H2O2(100 μmol/L) for 4 hours. MTT assay was used to analyze cardiomyocyte activity. Live/Dead viability/cytotoxicity kit was employed to evaluate survival of myocardial cells. The Electron microscopy, TUNEL and Live/Dead staining were employed to evaluate apoptotic morphological appearance of cardiomyocytes. The confocal laser scanning microscopy was performed to detect [Ca2+]i. The Bcl-2 and Caspase3 expression levels were quantified by Western blot. Results The MTT results showed that compared with the H2O2 group, the decline of myocardial cell viability in the ES-CM group was significantly suppressed(P<0.05). The test results of the cell viability/toxicity kit showed that compared with the H2O2 group, the decrease in the percentage of viable cells in the ES-CM group was significantly inhibited(P<0.05). The TUNEL apoptosis kit test results showed that compared with the H2O2 group, the increase in the percentage of cell apoptosis in the ES-CM group was significantly inhibited(P<0.05). The data of confocal microscopy showed that compared with the H2O2 group, the increase of [Ca2+]i intensity in the ES-CM group was significantly inhibited(P<0.05). Western blot results showed that compared with the H2O2 group, the expression of Bcl-2 protein in the ES-CM group was significantly increased(P<0.05), and the expression of Caspase3 protein was significantly reduced(P<0.05), but were unchanged in ES-M cultured cardiomyocytes(P>0.05). Conclusion The ES-CM inhibits cardiomyocyte apoptosis induced by H2O2 via regulating Bcl-2 and Caspase3 mediated signaling pathway.