下调CopineⅠ抑制缺氧/复氧诱导的H9c2细胞凋亡  

Depletion of copine-Ⅰ inhibits cell apoptosis induced by hypoxia/reoxygenation in H9c2 cells

作　　者：高群[1] 刘媛媛[1] 柏丹娜[1] 

机构地区：[1]解放军第323医院干一科,陕西西安710054

出　　处：《心脏杂志》2017年第3期264-268,共5页Chinese Heart Journal

摘　　要：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。AIM To determine the effects and mechanisms of copine-I （ CPNE1 ） on apoptosis induced by hypoxia/reoxygenation （H/R） in H9c2. METHODS H9c2 myocytes were subjected to H/R （24 11/ 6 h） and divided into control group （CON）, H/R group, negative control （NC） ＋ H/R and CPNE1 siRNA ＋ H/R group. H9e2 myocytes were pretreated with NF-KB inhibitor PDTC （ 10 p.M） for 30 rain to determine the potential mechanisms of CPNE1 in cell apoptosis. CPNE1 expression was detected using RT-PCR and Western blot. Activity of LDH was assayed by ELISA and cell apoptosis was measured using flow cytometry after staining with annexin V/P1. Western blot was utilized to detect the expressions of cleaved-caspase 3 （c-caspase3） , Bax, Bcl-2 and NF-KB and ELISA was used to determine the activity of NF-KB. P-~ESULTS Compared with CON group, mRNA and protein levels of CPNE1 in the H/R group increased. Moreover, H/R resulted in a significant increase in apoptosis rate, activity of LDH, and protein expressions of c-caspase3 and Bax. In addition, the anti-apoptotic protein Bel-2 was decreased. Depletion of CPNE] decreased the activity of LDH, apoptosis rate, and expressions of e-caspase3 and Bax but augmented Bcl-2 expression. Silencing of CPNE1 enhanced the activity and expression of NF-kB in the nucleus. Regulation of CPNE1 in cell apoptosis was abolished partly by PDTC. CONCLUSIONS CPNE1 silencing inhibits cell apoptosis induced by H/R in H9c2. The possible mechanism for a myocardial protective role of CPNE siRNA is enhancin~ the activity of NF-kB.

关 键 词：H9C2 copineⅠ 缺氧复氧 细胞凋亡 

分 类 号：Q26[生物学—细胞生物学]