质粒短发夹RNA对小鼠肾内髓集合管细胞Klotho基因的影响

Effect of Plasmid-mediated Short Hairpin RNA on the Klotho Gene in Medullary Collecting Duct Cells in Mice

目的筛选质粒,研究质粒短发夹RNA(sh RNA)对小鼠肾内髓集合管细胞(IMCD3)Klotho基因的抑制作用。方法针对Klotho基因的3个位点设计并合成3对Klotho-sh RNA序列(sh RNA转染1、2、3组),构建其表达质粒p RNAU6-Klotho,并设置阴性序列(阴性对照组)及正常IMCD3细胞(未处理组)为对照,以Lipofactine2000转染至IMCD3细胞,24 h后采用逆转录-聚合酶链反应(RT-PCR)及蛋白质印迹法检测IMCD3细胞Klotho基因m RNA及蛋白表达水平。结果转染后,RT-PCR显示sh RNA转染2组IMCD3细胞Klotho m RNA水平与未处理组和阴性对照组相比较,均存在统计学意义(P<0.01);蛋白质印迹法结果显示与未处理组、阴性对照组、sh RNA转染1组及sh RNA转染3组相比,sh RNA转染2组IMCD3细胞的Klotho蛋白水平差异均有统计学意义(P<0.001)。结论质粒sh RNA能高效抑制小鼠肾脏IMCD3细胞Klotho的表达,提示其在肾脏病研究中可有一定的作用,并为下一步研究筛选出一条合适的干扰序列。

Objective To assess the effect of plasmid-mediated short hairpin RNA（shRNA） on Klotho gene in mice medullary collecting duct（IMCD3） cells.Methods Three pairs of shRNA for Klotho（the first,second,and third pairs of shRNA） were designed and pRNAU6-Klotho were constructed,which were transfected into IMCD3 cells by Lipofactine2000.The negative control group and untreated group were set up at the same time.After 24 hours,the expressions of Klotho mRNA and protein levels were detected by reverse transcription- polymerase chain reaction（RTPCR） and Western blotting,respectively.Results The second pairs of shRNA had the best interference effect compared with the control group according to RT-PCR（P〈 0.01）.The results of Western blotting showed that the Klotho protein levels in the second pairs of shRNA group differed much from all the other 4 groups（P〈 0.001）.Conclusion Plasmamediated shRNA can highly inhibit the expression of Klotho,which suggests that it may be potential to study the pathogenesis in kidney disease.