摘要
目的研究在大鼠肾小管上皮细胞(NRK-52E)凋亡模型中Omi/HtrA2的表达,并观察抑制Omi/HtrA2表达后细胞凋亡的变化。方法用脂质体介导的基因转染方法将Omi/HtrA2的特异性shRNA表达载体251(Pgenesil-1/Omi/HtrA1 shRNA1)和252(Pgenesil-1/Omi/HtrA2 shRNA2)及阴性质粒HK(Pgenesil-1/HK)分别转入NRK-52E。应用Western印迹方法检测Omi/HtrA2及caspase3的表达,用DNA ladder检测各组细胞发生凋亡的情况。结果带有绿色荧光的NRK-52E即为成功转染细胞;在Western blot结果显示:对照组在缺氧复氧后Omi/HtrA2表达较正常组明显增强,而在转251和252组Omi/HtrA2表达较对照组减弱(P<0.05),但251和252两组间差异无统计学意义。对照组caspase3的表达明显较正常组增强(P<0.05)。且在Omi/HtrA2被抑制的两组caspase3的明显较对照组(P<0.05)。DNA ladder也显示:在对照组出现典型的DNA条带。251和252组条带减弱。结论通过RNA干扰技术成功抑制了Omi/HtrA2缺氧/复氧模型中Omi/HtrA2的表达,从而抑制了凋亡的发生。
Objective To investigate the protein expression of Omi/HtrA2 in NRK-52E apoptosis models and to observe the change of apoptosis after Omi/HtrA2 was inhibited. Methods The Omi/HtrA2-shRNA expression plasraids 251 （Pgenesil-1/Omi/HtrA1 shRNA1）, 252 （Pgenesil-1/Omi/HtrA2 shRNA2）, and negative plasmid HK （Pgene- sil-1/HK） were transfeeted into NRK-52E by liposome-mediated method. Protein expression of Omi/HtrA2 and caspase3 were quantified by Western blotting. Apoptosis in every group was analyzed by DNA ladder. Results Successfully transfected cells were presented with green fluorescent NRK-52E. Western blotting showed higher expression of Omi/HtrA2 in the control group than in normal group. There was a significant reduction in Omi/HtrA2 expression in the groups transfeetecl with 251 and 252 than in the controls （P〈0.05）, while statistical difference between the groups transfected 251 and 252 was not found. The protein expression of caspase3 of the control group increased comparing to the normal （P〈0.05）. The protein expression of caspase3 in the groups with inhibited Omi/HtrA2 was lower than that in the controls （P〈0.05）. The distinctive ladder pattern was visible in the control group but appeared weaker in the 251 and 252 groups. Conclusion RNA interference technique was successfully used to inhibit the expression of Omi/ HtrA2 in the hypoxia-reoxygenation model, which resulted in mitigation of apoptosis.
出处
《中国药物与临床》
CAS
2009年第1期9-12,共4页
Chinese Remedies & Clinics
基金
山西省自然科学基金(28011078-1)
山西医科大学2007年博士启动基金
