沉默CrkL对缺氧/复氧诱导的心肌细胞凋亡和生存力的影响及其机制

Effect of CrkL silence on hypoxia/reoxygenation-induced apoptosis and survival inhibition in cardiomyocytes and the underlying mechanisms

目的探讨沉默CrkL对缺氧/复氧诱导的心肌细胞凋亡和生存力的影响及其机制。方法分别用空白试剂、阴性慢病毒和CrkL RNAi慢病毒(沉默CrkL mRNA)感染H9C2心肌细胞,并进行缺氧/复氧(H/R)干预。实验分为空白组、阴性病毒组、CrkL沉默组、空白+H/R组、阴性病毒+H/R组、CrkL沉默+H/R组。用RT-PCR法检测心肌细胞CrkL mRNA的表达,蛋白印迹分析法检测心肌细胞CrkL蛋白、p-ERK1/2蛋白表达,MTT法检测细胞的增殖率,流式细胞术检测细胞的凋亡率。结果 CrkL沉默组较空白组和阴性病毒组,CrkL沉默+H/R组较空白+H/R组和阴性病毒+H/R组CrkL mRNA、CrkL蛋白、p-ERK1/2蛋白、细胞增殖率均降低(P<0.05或P<0.01),细胞凋亡率均增加(P<0.01)。结论沉默CrkL能加重缺氧/复氧诱导的心肌细胞凋亡和生存力降低。该过程可能是由p-ERK1/2蛋白表达降低介导的。

Objective To investigate the effect of CrkL silence on hypoxia/reoxygenation（H/R）-induced apoptosis and survival inhibition in cardiomyocytes and the underlying mechanisms.Methods H9C2 cardiomyocytes were infected with blank,negative lentivirus and CrkL RNAi lentivirus（CrkL mRNA silence）,and then treated with H/R separately. The cardiomyocytes were divided into blank group,negative lentivirus group,CrkL silence group,blank ＋ H/R group,negative lentivirus＋ H/R group and CrkL silence＋ H/R group.The expression of CrkL mRNA was detected by RT-PCR,and the expression of CrkL and p-ERK1/2protein was detected by Western blotting analysis.The apoptosis rate of cardiomyocytes was analyzed by flow cytometry,and the cell proliferation rate was analyzed by MTT.Results Compared with blank and negative lentivirus groups,CrkL silence group had significantly decreased CrkL mRNA and protein,p-ERK1 protein,p-ERK1/2protein and proliferation rate（P〈0.05 or P〈0.01）,and significantly increased apoptosis rate（P〈0.01）.The same was true for CrkL silence＋ H/R group when compared with the blank＋ H/R and negative lentivirus＋ H/R groups.Conclusion CrkL silence can aggravate H/R-induced apoptosis and survival inhibition in cardiomyocytes,which might be mediated by the decrease of pERK1/2protein expression.