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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 study the effect of small interfering RNA (siRNA) on the expression of Ppif gene in rat and to explore its effect on the proliferation and apoptosis of rat renal cell (NRK). Methods: siRNA targeting Ppif gene was synthesized in vitro and transfected into NRK cells by lipofectamine. The expression of Ppif mRNA was detected by RT-PCR. The expression of Ppif protein was detected by Western blot. The apoptosis of cells was detected by flow cytometry MTT assay was used to detect cell proliferation activity. RESULTS: After 24 h of Ppif siRNA transfection, compared with the control group, three siRNAs targeting different targets had no significant inhibitory effect on Ppif at the gene level (P > 0.05). The siRNA at the 198 site showed a significant inhibitory effect on gene and protein levels, with a 75.25% decrease in gene level and a 72.13% decrease in protein levels (P <0.05). The MTT results showed that the proliferation of NRK cells was significantly increased, and the inhibitory rates of siRNA1 ~ siRNA4 were (68.6 ± 3.6)%, (5.3 ± 4.3)%, (7.6 ± 6.3)% and (4.1 ± 4.5) %, Apoptosis rate decreased significantly. CONCLUSION: Ppif siRNA synthesized in vitro (starting at 198) can effectively inhibit the expression of Ppif in NRK cells, thereby inhibiting cell apoptosis and promoting cell proliferation.