shRNA沉默ROCK1和ROCK2表达对缺氧诱导的大鼠心肌细胞凋亡的影响  

Effect of ROCK1and ROCK2silencing by shRNA on hypoxia-induced apoptosis of rat cardiomyocytes

作　　者：孙国芳[1] 丁浩[1] 李菊香[1] 洪葵[1] 董家龙[1] 吴清华[1] 程晓曙[1] 

机构地区：[1]南昌大学第二附属医院心内科,江西省分子医学重点实验室,江西南昌330006

出　　处：《中国病理生理杂志》2011年第12期2307-2312,共6页Chinese Journal of Pathophysiology

基　　金：国家重点基础研究发展计划(“973”计划)(No.2008CB517305);国家自然科学基金资助项目(No.81060013)

摘　　要：目的:探讨Rho相关的卷曲蛋白激酶(ROCK1)和ROCK2在缺氧诱导的大鼠心肌细胞凋亡中的作用。方法:原代培养大鼠心肌细胞,并用抗α-横纹肌肌动蛋白免疫组化法进行鉴定。将ROCK1-shRNA和ROCK2-shRNA瞬时转染细胞,48 h后给予缺氧6 h处理。实验分为5组:(1)空白对照组;(2)缺氧组;(3)缺氧+阴性对照shRNA组;(4)缺氧+ROCK1-shRNA组;(5)缺氧+ROCK2-shRNA组。用倒置显微镜观察心肌细胞搏动频率与节律;用全自动生化分析仪测定细胞培养液中乳酸脱氢酶(LDH)含量;用MTT检测细胞存活率;用流式细胞仪检测细胞凋亡率;用Western blotting检测ROCK1和ROCK2的表达,并检测caspase-3和p-PI3K的表达情况。结果:鉴定证实大鼠心肌细胞原代培养成功。缺氧损伤后心肌细胞搏动频率较对照组明显减慢,搏动幅度减弱,节律不规整(P<0.01),而ROCK1-shRNA和ROCK2-shRNA的转染能抑制缺氧导致的这一作用(P<0.05或P<0.01)。全自动生化分析仪检测发现,缺氧能导致细胞培养液LDH含量升高(P<0.01),而ROCK1-shRNA和ROCK2-shRNA的转染能抑制缺氧导致的这一作用(P<0.01)。MTT检测发现,缺氧能导致心肌细胞存活率降低(P<0.05),而ROCK1-shRNA和ROCK2-shRNA的转染能抑制缺氧导致的这一作用(P<0.05)。流式细胞仪检测发现,缺氧能导致心肌细胞凋亡率升高(P<0.01),而ROCK1-shRNA和ROCK2-shRNA的转染能抑制缺氧导致的这一作用(P<0.05或P<0.01)。Western blotting检测发现,ROCK1-shRNA的转染能降低ROCK1的表达(P<0.05),ROCK2-shRNA的转染能降低ROCK2的表达(P<0.01);缺氧能导致caspase-3表达升高(P<0.05)、p-PI3K表达降低(P<0.01),ROCK1-shRNA和ROCK2-shRNA的转染能抑制缺氧导致的这一作用(P<0.05或P<0.01)。结论:ROCK1和ROCK2表达下调能抑制缺氧损伤导致的大鼠心肌细胞凋亡,其机制与抑制caspase-3活化和增强p-PI3K的表达有关。AIM： To investigate the effects of Rho-associated coiled-coil protein kinase-1（ROCK1） and ROCK2 on apoptosis induced by hypoxia in rat cardiomyocytes.METHODS： Rat cardiomyocytes were cultured primarily and identified using an antibody targeting α-actin of striated muscle.ROCK1-shRNA and ROCK2-shRNA were transiently transfected into the cells by liposome.After 48 h,these cells were subject to hypoxia for 6 h.The cells were divided into 5 groups： blank control group,hypoxia group,hypoxia＋negative control shRNA group,hypoxia＋ROCK1-shRNA group and hypoxia＋ROCK2-shRNA group.The beating frequency and rhythm of the cardiomyocytes were assessed by microscopy.The activity of lactate dehydrogenase（LDH） in the cell culture supernatants was detected by automatic biochemical analyzer.The cell survival rate was analyzed by the method of MTT.The cell apoptotic rate was assessed by flow cytometry.Western blotting was used to determine the expression of ROCK1,ROCK2,caspase-3 and p-PI3K.RESULTS： The primary culture of the cardiomyocytes was successful.Western blotting results showed that the transfection of ROCK1-shRNA or ROCK2-shRNA decreased the expression of ROCK1 or ROCK2 in the cardiomyocytes.Hypoxia slowed down the beat frequency of the cardiomyocytes,also made the rhythm disorder.Hypoxia increased the release of LDH and decreased the cell survival rate.Flow cytometry results showed that hypoxia increased the cell apoptotic rate.Hypoxia increased the expression of caspase-3 and decreased the expression of p-PI3K.Transfection of ROCK1-shRNA and ROCK2-shRNA into the cardiomyocytes reduced all the effects of hypoxia mentioned above.CONCLUSION： Down-regulation of ROCK1 and ROCK2 expression suppresses the apoptosis of rat cardiomyocytes induced by hypoxia.The mechanism is associated with the inhibition of caspase-3 activation and the up-regulation of p-PI3K expression.

关 键 词：Rho相关的卷曲蛋白激酶 心肌细胞 SHRNA 缺氧 细胞凋亡 

分 类 号：R363[医药卫生—病理学]