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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 screen plasmids and study the inhibitory effect of plasmid short hairpin RNA (shRNA) on Klotho gene in mouse renal medullary collecting duct cells (IMCD3). Methods Three pairs of Klotho-sh RNA sequences were designed and synthesized based on 3 sites of Klotho gene (sh RNA were transfected into groups 1, 2, and 3). The expression vector p RNAU6-Klotho was constructed and negative sequence was set (negative control group) IMCD3 cells (untreated group) were used as control and transfected into IMCD3 cells with Lipofactine2000. The expression of m RNA and protein of Klotho gene in IMCD3 cells was detected by RT-PCR and Western blot 24 hours later Level. Results After transfection, RT-PCR showed that the Klotho m RNA level of sh RNA transfected two groups of IMCD3 cells was significantly higher than that of untreated group and negative control group (P <0.01) There were significant differences in Klotho protein levels between sh RNA transfected two groups of IMCD3 cells (P <0.001) compared with the control group, the negative control group, sh RNA transfected group 1 and sh RNA transfected 3 groups. Conclusion Plasmid sh RNA can effectively inhibit the expression of Klotho in mouse renal IMCD3 cells, suggesting that it may play a role in the study of kidney disease and screen a suitable interference sequence for further study.