小干扰RNA沉默HK-2细胞VDR及其对低枸橼酸尿症的意义

Small Interfering RNA Silencing VDR in HK-2 Cells and Its Significance for Hypocitraturia

目的筛选有效沉默人肾近曲小管上皮细胞(HK-2 cells,HK-2细胞)维生素D受体(vitamin D receptor,VDR)基因的小干扰核糖核酸(small interfering ribonucleic acid,si RNA)及其对低枸橼酸尿症的意义.方法培养HK-2细胞,设计4条VDR-si RNA,利用包装的慢病毒侵染所培养的HK-2细胞,利用蛋白质印迹法(western blot,WB)和实时定量聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)检测沉默VDR基因的HK-2细胞中VDR蛋白质和VDR-m RNA的表达水平.结果 si RNA 1(6120)能显著抑制HK-2细胞中VDR的表达,获得了VDR-si RNA 1(6120)稳定细胞株.结论 VDR-si RNA 1(6120)能有效沉默HK-2细胞中VDR的表达,可以进行下一步沉默VDR表达后检测Na(+)-二羧酸转运子[Na(+)-dicarboxylate cotransporter1,Na DC1]表达情况.

Objective To screen small interfering ribonucleic acid(siRNA)that can effectively silence the vitamin D receptor(VDR)of human renal proximal tubular epithelial cells(HK-2cells)and its significance for hypocitraturia.Methods We cultured HK-2cells,designed4VDR-siRNA,used packaged lentivirus to infect HK-2cells and detected the expression level of VDR protein and mRNA of HK-2cells whose VDR gene silenced by Western blot and real-time quantitative PCR.Results SiRNA1(6120)could significantly inhibit the expression of VDR in HK-2cells,and siRNA VDR-1(6120)stable cell lines were obtained.Conclusion VDR-siRNA1(6120)can effectively silence the expression of VDR in HK-2cells,and we can detect the expression of NaDC1after VDR expression is inhibited in the next step.