论文部分内容阅读
目的研究CopineⅠ(CPNE1)对缺氧/复氧(Hypoxia/Reoxygenation,H/R)诱导H9c2细胞凋亡的作用及可能的机制。方法以H9c2心肌细胞为研究对象,建立H/R模型,细胞被随机分为对照组(CON)、H/R组、阴性对照(NC)+H/R和CPNE1 siRNA+H/R组,阻断实验用NF-κB的阻断剂PDTC(10μmol/L)预处理细胞30 min。RT-PCR和Western blot方法用于检测CPNE1表达水平。细胞乳酸脱氢酶(LDH)活性采用ELISA方法检测。细胞经AnnexinV/PI染色后用流式细胞仪检测凋亡率,Western blot方法检测claved-caspase3(c-caspase3)、Bcl-2和Bax的蛋白表达水平。细胞中NF-κB活性采用ELISA方法检测。结果与CON组相比,H/R组CPNE1表达水平上调、LDH活性升高、凋亡率上升、c-caspase3和Bax蛋白表达升高、Bcl-2蛋白表达水平下降。与NC+H/R组相比,CPNE1 siRNA+H/R组细胞的LDH活性下降、凋亡率降低、c-caspase3和Bax蛋白表达减少、Bcl-2表达增多。此外,沉默CPNE1细胞核中NF-κB活性增强且蛋白表达上升,PDTC可逆转CPNE1 siRNA对细胞凋亡的抑制作用。结论下调CPNE1的表达能够抑制H/R诱导的H9c2细胞凋亡,其可能机制是通过增强NF-κB活性发挥心肌保护作用。
Objective To investigate the effect and possible mechanism of copine Ⅰ (CPNE1) on H9c2 cell apoptosis induced by hypoxia / reoxygenation (H / R). Methods H9c2 cardiomyocytes were used to establish H / R model. The cells were randomly divided into control group (CON), H / R group, negative control (NC) + H / R and CPNE1 siRNA + H / R group The experiment was pretreated with NF-κB blocker PDTC (10μmol / L) for 30 min. RT-PCR and Western blot were used to detect the expression of CPNE1. Cell lactate dehydrogenase (LDH) activity was measured by ELISA. The apoptotic rate was detected by flow cytometry after AnnexinV / PI staining. The protein expression of claved-caspase3, Bcl-2 and Bax were detected by Western blot. The activity of NF-κB in cells was detected by ELISA. Results Compared with CON group, the expression of CPNE1 in H / R group increased, the activity of LDH increased, the apoptosis rate increased, the expressions of c-caspase3 and Bax protein increased and the expression of Bcl-2 protein decreased. Compared with NC + H / R group, the activity of LDH decreased, the apoptosis rate decreased, the expression of c-caspase3 and Bax decreased and the expression of Bcl-2 increased in CPNE1 siRNA + H / R group. In addition, NF-κB activation and protein expression were increased in the silenced CPNE1 cells. PDTC could reverse the inhibitory effect of CPNE1 siRNA on apoptosis. Conclusion Down-regulation of CPNE1 can inhibit H / R-induced apoptosis of H9c2 cells, and its possible mechanism is to exert myocardial protective effect by enhancing NF-κB activity.