生长停滞特异性基因6对大鼠心肌细胞缺血再灌注的保护机制

摘要

目的 初步探讨生长停滞特异性基因6(Gas6)在缺血预适应(IPC)中,对大鼠心肌细胞缺氧/复氧(I/R)损伤的保护作用及机制.方法 选择大鼠胚胎源性H9C2心肌细胞株培养48 h后,随机分为5组:NC组、I/R组、IPC组、Gas6组、Gas6+ LY组为Gas6+磷脂酰肌醇3激酶(PI3K)阻断剂LY294002培养,各组按分组条件培养后,测乳酸脱氢酶(LDH)活性；流式细胞术测细胞凋亡率；实时荧光定量PCR法测NC组、I/R组和IPC组Gas6 mRNA表达；Western blot法测磷酸化蛋白激酶B(p-Akt),磷酸化叉头蛋白O亚家族(p-Foxo3a)蛋白水平.结果 与NC组比较,I/R组LDH活性和细胞凋亡率明显升高；与I/R组比较,IPC组Gas6 mRNA表达增加,IPC组和Gas6组LDH活性及细胞凋亡率明显减少,p-Akt、p-Foxo3a蛋白明显增加,与Gas6组比较,Gas6+LY组LDH活性和细胞凋亡率明显升高,p-Akt和p-Foxo3a蛋白表达明显下降(P＜0.05).结论 Gas6通过PI3K/Akt/Foxo3a通路在IPC中起心肌细胞保护作用.%Objective to study the protective effect and mechanism of Gas6-induced cardioprotection with ischemic preconditioning mediated by the rat embryonic heart-derived cell line,H9C2 cells. Methods The H9C2 cells were randomly divided into five groups: normal control, IR, ischemic pre-conditioning(IPC) ,Gas6 and Gas6 + LY294002 group. Serum LDH was detected with kit;cell apoptotic rate was evaluated by flow cytometry; the Gas6 mRNA was examined by the real-time PCR,and the protein expressions of p-Akt and p-Foxo3a were examined by Western blot. ResultsCompared with I/R group, Gas6 mRNA expression in IPC group was significantly increased (P<0. 05),serum LDH level and the cell apoptotic rate were significantly decreased in IPC group and Gas6 group(P<0. 05), meanwhile protein expressions of p-Akt and p-Foxo3a were significantly increased(P<0. 05) ;in IPC group serum LDH level,the cell apoptotic rate and protein expressions of p-Akt and p-Foxo3a were not different from that in Gas6 group(P>0. 05) ,but serum LDH level and the cell apoptotic rate were significantly increased in Gas6 + LY294002 group, meanwhile p-Akt and p-Foxo3a protein expressions significantly decreased(P<0. 05). ConclusionGas6 showed a protective effect in ischemia preconditioning, that may be mediated by Phos-phatidylinositol3-Kinase/Akt/Foxo3a Survival Pathway.