CAJ | 학술논문

目的构建干扰Ppif基因的重组U6慢病毒质粒,观察内源性小干扰RNA(siRNA)的效果.方法根据siRNA设计原则,设计合成短发卡RNA(shRNA),用于体内转录合成干扰Ppif编码序列的发夹RNA.连接、筛选、酶切,鉴定Pgcl-GFP/U6 Ppif shRNA;以含无关序列发夹结构的质粒载体为对照,感染大鼠肾细胞株,采用实时定量聚合酶链反应(Real-time PCR)和Western blot检测mRNA和蛋白表达水平与对照组的差异.结果测序证实重组质粒构建成功,体外试验表明,Ppif基因的mRNA和蛋白表达均一致降低[(0.582 ±0.014)％],与阴性对照组[(0.946±0.018)％]和空白对照组[(0.893±0.021)％]比较,差异有统计学意义(P＜0.05).结论慢病毒介导的Ppif-shRNA成功在体外敲减了大鼠肾细胞的目的蛋白表达,影响了肾缺血再灌注的发生与发展.
목적 구건간우Ppif기인적중조U6만병독질립,관찰내원성소간우RNA(siRNA)적효과.방법 근거siRNA설계원칙,설계합성단발잡RNA(shRNA),용우체내전록합성간우Ppif편마서렬적발협RNA.련접、사선、매절,감정Pgcl-GFP/U6 Ppif shRNA;이함무관서렬발협결구적질립재체위대조,감염대서신세포주,채용실시정량취합매련반응(Real-time PCR)화Western blot검측mRNA화단백표체수평여대조조적차이.결과 측서증실중조질립구건성공,체외시험표명,Ppif기인적mRNA화단백표체균일치강저[(0.582 ±0.014)％],여음성대조조[(0.946±0.018)％]화공백대조조[(0.893±0.021)％]비교,차이유통계학의의(P＜0.05).결론 만병독개도적Ppif-shRNA성공재체외고감료대서신세포적목적단백표체,영향료신결혈재관주적발생여발전.
Objective To construct a recombinant lentiviral U6 plasmids of RNA interference (RNAi) of Ppif gene,and evaluate and observe the interference effects of Ppif-short hairpin RNA (shRNA)/U6 vector on normal rat kidney (NRK) cells by endogenous small RNAi in vitro.Methods According to the target sequence and design principle of siRNA,the short hairp in RNA was designed and synthesized to interfere with Ppif gene.The shDNA duplex was ligated into the recombinant vector pGCLGFP/U6 plasmid.Renal cells were transfected with pGCL-GFP/U6 Ppif shRNA,and pGCL-GFP/U6 scrambled shDNA served as control.The expression level of Ppif was detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.Results The recombinant vector was successfully constructed and confirmed by sequencing.The in vitro experiment indicated that the expression level of Ppif protein and mRNA was down-regulated [(0.582 ±0.014) ％] in comparison to negative control group [(0.946 ± 0.018) ％] and blank control group [(0.893 ± 0.021) ％] (P ＜ 0.05 for all).Conclusion RNA interference can successfully down-regulate the expression of Ppif on NRK cells and may affect the occurrence and development of renal ischemia-reperfusion injury.