Omi/HtrA2短发夹RNA在大鼠肾小管上皮细胞凋亡中的作用及机制

目的探讨Omi/HtrA2短发夹RNA(shRNA)对缺氧/复氧诱导大鼠肾小管上皮细胞(NRK-52E)凋亡的作用及机制。方法细胞分为5组:正常组(常规培养),模型组(缺氧/复氧组)、HK组(转染重组质粒Pgenesil-1/HK)、shRNA1组(转染Pgenesil-1/Omi/HtrA2shRNA1)、shRNA2组(转染Pgenesil-1/Omi/HtrA2shRNA2)。用荧光显微镜观察荧光蛋白的表达,Westernblot检测各组Omi/HtrA2、半胱氨酰天冬氨酸特异性蛋白酶(caspase)-3/-9蛋白表达,比色法测定caspase-3/-9的活性。结果在荧光显微镜下,转染组细胞均可见绿色荧光,未转染组未见绿色荧光。与模型组相比,shRNA1组和shRNA2组Omi/HtrA2、caspase-3/-9蛋白表达明显减少(P<0.01)。模型组和HK组、shRNA1组和shRNA2组之间Omi/HtrA2、caspase-3/-9蛋白表达差异无统计学意义。与正常组相比,模型组Omi/HtrA2、caspase-3/-9蛋白表达明显增强(P<0.01)。结论Pgenesil-1/Omi/HtrA2shRNA1和Pgenesil-1/Omi/HtrA2shRNA2能明显减少缺氧/复氧诱导的NRK-52E中Omi/HtrA2蛋白的表达。通过抑制procaspase-9的活化,进而减少了下游procaspase-3的激活,最终减轻了缺氧/复氧诱导的NRK-52E的凋亡程度。

Mechanisms and effects of Omi/HtrA2 shRNA on the apoptosis of rat renal tubular epithelial cells

Objective To investigate the mechanisms and effects of Omi/HtrA2 short hairpin RNA （shRNA） on the apoptosis of rat renal tubular epithelial cells （NRK-52E） as a result from hypoxia-reoxygenation damage. Methods The cells were divided into 5 groups： normal group （cultured under routine condition）, model group （subjected to hypoxia-reoxygenation）, HK group （transfected with Pgenesil-1/HK）, shRNA1 group （transfected with Pgenesil-1/Omi/ HtrA2 shRNA1）, and shRNA2 group （transfected with Pgenesil-1/Omi/HtrA2 shRNA2）. The fluorescent microscopy was used to examine the expression of fluorescent protein. The expressions of Omi/HtrA2 and caspase-3/-9 were examined by Western blotting and the activity of caspase-3/-9 was analyzed by colorimetry. Results The expression of fluorescent protein was detected in transfected cells but not in nontransfected ones. Compared to model group, the expressions of Omi/HtrA2 and caspase-3/-9 were significantly decreased in shRNA1 and shRNA2 groups （P〈0.01）. Compared to normal groups, the expressions of Omi/HtrA2 and caspase-3/-9 were obviously increased in model group （P〈 0.01）. The expressions of Omi/HtrA2 and caspase-3/-9 did not differ either between model group and HK group or between shRNA1 group and shRNA2 group. The activity of caspase-3/-9 well correlated with the expressions of caspase-3/-9 in all groups. Conclusion The Pgenesil-1/Omi/HtrA2 shRNA1 and Pgenesil-1/Omi/HtrA2 shRNA2 may markedly inhibit the expressions of Omi/HtrA2 in NRK-52E. Apoptosis of NRK-52E from hypoxia-reoxygenation injury was shown to be inhibited through blockage of caspase-9 activation and subsequently of caspase-3.