This work
was supported by the National Natural Science Foundation of China (No. 30100069).
*Corresponding auther.
Tel: +86-23- 68774859; E-mail: fxb12@yahoo.com.cn
凋亡诱导因子介导缺氧/复氧致肥大心肌细胞凋亡的作用
冯兵*，周小波，杨 旭，叶自林，何作云
第三军医大学新桥医院肾内科，重庆 400037
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-specific
cysteinyl proteinase, 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-inducing factor, 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（0.1
mmol/L培养12
h） 诱导培养的小鼠心肌细胞肥大，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、Western blot、siRNA基因转染、Hoechst 33258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) AIF mRNA及蛋白表达水平均显著升高（mRNA：0.52±0.04 及0.85±0.10 vs
0.29±0.08，P<0.05；蛋白质：2.07±0.15和3.12±0.19
vs 0.29±0.04，P<0.05），即随缺血时间的延长，AIF mRNA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AIF
mRNA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) AIF siRNA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AIF
mRNA、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。
关键词： 心肌肥大；细胞凋亡；缺氧/复氧；AIF
Apoptosis of
hypertrophic cardiomyocytes
stimulated by hypoxia- reoxygenation is partially
mediated by apoptosis-inducing factor
FENG Bing*, ZHOU Xiao-Bo, YANG Xu,
YE Zi-Ling, He Zuo-Yun.
Department of Nephrology,
Abstract:
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 apoptosis-inducing 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 still not understood. The purpose
of this study was to evaluate hypoxia-reperfusion-induced alterations of AIF
mRNA and protein expression in hypertrophic cardiomyocytes. Cardiomyocyte
hypertrophy was produced by angiotensin II (0.1 mmol/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 stainning.
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 of AIF mRNA expression was 0.29±0.08 (optical
density, relative value) in the control group (hypertrophic cardiomyocytes
cultured under 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±
Key
words: cardiac
hypertrophy; apoptosis; hypoxia-reoxygenation; apoptosis-inducing
factor