凋亡诱导因子介导缺氧/复氧致肥大心肌细胞凋亡的作用  

Apoptosis of hypertrophic cardiomyocytes stimulated by hypoxia-reoxygenation is partially mediated by apoptosis-inducing factor

作　　者：冯兵[1] 周小波[1] 杨旭[1] 叶自林[1] 何作云[1] 

机构地区：[1]第三军医大学新桥医院肾内科,重庆400037

出　　处：《生理学报》2006年第6期599-605,共7页Acta Physiologica Sinica

基　　金：This work was supported by the National Natural Science Foundation of China (No. 30100069).

摘　　要：心肌细胞凋亡导致心肌组织合胞体功能丧失,最终使代偿性心肌肥大向心力衰竭转化。过去的研究已经确认天门冬氨酸特异性半胱氨酸蛋白酶(caspartate-specificcysteinylproteinase,caspase)依赖机制在心肌细胞凋亡中的作用,但对caspase非依赖机制即凋亡诱导因子(apoptosis-inducingfactor,AIF)在心肌细胞凋亡中的作用尚不明确。本研究应用血管紧张素Ⅱ(0.1μmol/L培养12h)诱导培养的小鼠肥大心肌细胞,利用三气孵箱建立缺氧/复氧模型以模拟缺血再灌注。应用RT-PCR、Westernblot、siRNA基因转染、Hoechst33258染色法检测AIF在mRNA和蛋白质水平的表达及细胞凋亡的变化,分析AIF在缺氧/复氧致肥大心肌细胞凋亡中的意义。结果如下:(1)与对照组比较,缺氧8h组(H8h)和缺氧12h组(H12h)AIFmRNA及蛋白表达水平均显著升高(mRNA:0.52±0.04及0.85±0.10vs0.29±0.08,P<0.05;蛋白质:2.07±0.15和3.12±0.19vs0.29±0.04,P<0.05),即随缺血时间的延长,AIFmRNA及蛋白表达水平均显著增加。(2)与对应单纯缺氧组比较,缺氧后给予复氧刺激,H8h/R组和H12h/R组AIFmRNA及蛋白表达水平均显著升高(mRNA:1.09±0.12和1.41±0.23,P<0.05;蛋白质:4.57±0.25和5.71±0.27,P<0.05)。仅在H8h/R及H12h/R组,可见AIF核转位显著增加。(3)AIFsiRNA转染可显著抑制肥大心肌细胞AIF的表达,对缺氧时细胞凋亡无明显影响(P>0.05),但可显著降低缺氧/复氧诱导的肥大心肌细胞凋亡率(P<0.05)。同时抑制AIF及caspase-3活性,可显著加强单一抑制剂对缺氧/复氧诱导的肥大心肌细胞凋亡的抑制作用。(4)抑制caspase-3活性对缺氧/复氧诱导的AIF核转位无明显影响。上述结果提示,缺氧/复氧时AIFmRNA、蛋白表达和核转位均显著增加,且在缺氧/复氧诱导肥大心肌细胞凋亡中具有重要的作用。Cardiomyocyte apoptosis leads to the functional incapacitation of myocardial plasmodium and plays an important role in the pathogenesis of heart failure transformed from compensable cardiac hypertrophy. Mitochondria are the main source of apoptosisinducing molecule of various cells, and the role of caspartate-specific cysteinyl proteinase （caspase）-dependent mechanism has generally been accepted in the cardiomyocyte apoptosis. However, the significance of caspase-independent apoptosis-inducing factor （AIF） mechanism is not yet understood. The purpose of this study was to evaluate hypoxia-reperfusion-induced alterations of AIF mRNA and protein expressions in hypertrophic cardiomyocytes. Cardiomyocyte hypertrophy was produced by angiotensin Ⅱ （0.1 μmol/L）. The cells were cultured under the condition of hypoxia （95% N2 and 5% CO2; the O2 partial pressure was lower than 5 mmHg） for 8 h or 12 h （named as H8h and H12h groups, respectively）, and then exposed to normal culture environment （named as H8h/R and H12h/R groups, respectively）. Apoptosis was detected with Hoechst 33258 staining. The AIF mRNA and protein expressions were detected by RT-PCR and Western blot and quantified by gel scanning. The results were as follows： （1） The level ofAIF mRNA expression was 0.29±0.08 （optical density, relative value） in the control group （hypertrophic cardiomyocytes cultured in normal environment）. Compared with that in the control group, the levels of AIF mRNA expression were significantly higher in the groups of H8h and H12h （0.52±0.04 and 0.85±0.10）, indicating that this effect was time-dependent. A further increase of AIF mRNA expression was observed in the groups of H8h/R （1.09±0.12） and H12h/R （1.41±0.23）. （2） The level of AIF protein expression was 0.29±0.04 in the control group. Compared with that in the control group, the levels of AIF protein expression were significantly higher in the groups of H8h and H12h （2.07±0.15 and 3.12±0.19）. The AIF protein ex

关 键 词：心肌肥大 细胞凋亡 缺氧/复氧 凋亡诱导因子 

分 类 号：R363[医药卫生—病理学]