摘要
目的:应用小干扰RNA(small interfering RNA,siRNA)抑制大鼠基因Ppif的表达,探讨其对大鼠肾细胞(NRK)增殖和凋亡的影响。方法:体外化学合成针对Ppif基因的siRNA序列,在脂质体介导下转染NRK细胞,RT-PCR检测Ppif mRNA表达水平,Western blot检测Ppif蛋白表达水平,流式细胞仪检测细胞凋亡状况,MTT法检测细胞增殖活性。结果:Ppif siRNA转染NRK细胞24 h后,所设计的3对针对不同靶点的siRNA与对照组相比,起始于405、556位点的siRNA在基因水平对Ppif无明显抑制效应(P>0.05);起始于198位点的siRNA在基因和蛋白水平均有明显的抑制效应,基因水平下降75.25%(P<0.05);蛋白水平下降72.13%(P<0.05)。MTT结果显示NRK细胞增殖能力显著增加,转染24 h后,siRNA1～siRNA4细胞抑制率分别为(68.6±3.6)%、(5.3±4.3)%、(7.6±6.3)%和(4.1±4.5)%,凋亡率明显下降。结论:体外化学合成的Ppif siRNA(起始于198位点)可以有效地抑制NRK细胞Ppif的表达,从而抑制细胞凋亡,促进细胞增殖。
Objective：To evaluate the effect of targeting Ppif gene on the proliferation and apoptosis of NRK cells. Methods：Chemically synthesized small interfering RNA （ siRNA ） targeting Ppif was transfeeted into NRK cell line NRK by LipofectamineTM2000. The expression levels of Ppif mRNA and protein were detected respectively by RT-PCR and Western blot. Cell proliferation was determined by MTT, and cell apoptosis was observed by flow cytometry. Results：At 24 hours after transfection,the siRNAs located in the 405 and 556 gene positions had no significant inhibition effect for Ppif （P〉0. 05）. However, the siRNA located in the 198 gene position significantly decreased the expression of Ppif at gene （decreased by 75.25%, P〈0.05） and protein levels （decreased by 72. 13%, P〈0.05）. MTT results showed that the growth rate of NRK cells was decreased markedly. In the groups of siRNA1, siRNA2, siRNA3 and siRNA4, the inhibition ratios after 24 hours transfection were （68.6± 3.6 ）%, （5.3 ±4.3）%. （ 7.6 ± 6.3）% and （4.1 ±4.5）, and the apoptosis was decreased significantly. Conclusions： The siRNA located in the 198 gene position could effectively inhibit the expression of Ppif in NRK cells, and inhibit cell apoptosis proliferation and induce cell proliferation.
出处
《临床泌尿外科杂志》
北大核心
2010年第1期53-57,共5页
Journal of Clinical Urology
基金
国家自然科学基金资助项目(No.30670820)
