中文摘要：

目的：通过建立体外心肌细胞缺氧模型,观察Rho相关的卷曲螺旋蛋白激酶（ROCK）在不同缺氧时点的表达,探讨其在心肌细胞凋亡中的作用。方法：（1）原代培养SD乳鼠心肌细胞,并用抗α-横纹肌肌动蛋白免疫组化法进行鉴定。（2）利用缺氧液和缺氧盒,建立心肌细胞缺氧模型,分别缺氧培养1 h、3 h、6 h、9 h,以正常氧培养的心肌细胞作为对照。（3）用流式细胞仪检测心肌细胞凋亡,MTT检测细胞存活率,Western blotting检测ROCK-1、ROCK-2、caspase-3活化片段、PI3K和p-PI3K的表达情况。结果：（1）缺氧1 h、3 h、6 h、9 h后,心肌细胞凋亡率分别为（8.76±1.51）%、（15.36±2.34）%、（26.50±3.43）%、（41.96±4.22）%,与对照组[2.60±0.34）%]比较均有显著差异（P〈0.01）;存活率分别为（93.20±4.12）%、（86.14±3.10）%、（75.53±7.25）%、（60.21±6.75）%,与对照组[（97.60±1.12）%]比较均有显著差异（P〈0.05）。（2）ROCK-1和ROCK-2缺氧1 h即开始升高,3-6 h表达达高峰,9 h开始回落,且均明显高于对照组（P〈0.05）。（3）Caspase-3在缺氧1 h开始逐渐活化,在随后观察的时点内持续处于活化状态,与对照组比较均有显著差异（P〈0.01）。（4）p-PI3K在缺氧1 h后开始升高,3 h达高峰,6 h开始回落,9 h表达基本消失,各时点比较差别有统计学意义（P〈0.05）。结论：ROCK-1和ROCK-2在缺氧后表达逐渐升高,与细胞凋亡的过程相一致。ROCK可能通过抑制p-PI3K信号途径,促进缺氧心肌细胞凋亡。

英文摘要：

AIM： To investigate the expression of Rho-associated coiled-coil protein kinase（ ROCK） at different hypoxic phases and to explore its role in myocardial cell apoptosis. METHODS： The rat cardiomyocytes were primarily cultured and identified by an antibody targeting α-actin of striated muscle. The myocardial cell hypoxic model was established by exposing the cells in hypoxic liquid for 1 h,3 h,6 h and 9 h. The cell apoptotic rate was assessed by flow cytometry. The cell survival rate was determined by MTT assay. The protein levels of ROCK-1,ROCK-2,caspase-3 activation fragment,PI3 K and p-PI3 K at different hypoxia phases were determined by Western blotting. RESULTS： After exposed to hypoxic liquid for 1 h,3 h,6 h and 9 h,the apoptotic rates of the cardiomyocytes were（ 8. 76 ± 1. 51） %,（ 15. 36 ±2. 34） %,（ 26. 50 ± 3. 43） % and（ 41. 96 ± 4. 22） %,respectively,significantly higher than those in control group[（ 2. 60 ± 0. 34） %,P〈0. 01]. The survival rates were（ 93. 20 ± 4. 12） %,（ 86. 14 ± 3. 10） %,（ 75. 53 ± 7. 25） % and（ 60. 21 ± 6. 75） %,respectively,signficantly lower than those in control group [（ 97. 60 ± 1. 12） %,P〈0. 05]. After 1 h of hypoxic exposure,the levels of ROCK-1 and ROCK-2 began to rise,reached its peak at 3 - 6 h,and began to decrease after 9 h,which were significantly higher than those in control group（ P〈0. 05）. After 1 h of hypoxic exposure,the caspase-3 activation fragment began to rise,which was sustained in a high level at following observed time points as compared with control group（ P〈0. 01）. No difference of the PI3 K expression in the course of hypoxia was observed. However,after 1 h of hypoxic exposure,the p-PI3 K level began to rise,reached its peak at 3 h,began to decrease at 6 h,and was almost undetectable at 9 h. CONCLUSION： Hypoxia stimulates the cardiomyocytes to increase the expression of ROCK-1 and ROCK-2,and is in parallel with the cardiomyocyte apoptosis. ROCKs may play an important role in the proc