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
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摘要: 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.Abstract: Objective To screen small interfering ribonucleic acid (si RNA) that can effectively silence the vitamin D receptor (VDR) of human renal proximal tubular epithelial cells (HK-2 cells) and its significance for hypocitraturia. Methods We cultured HK-2 cells, designed 4 VDR-si RNA, used packaged lentivirus to infect HK-2 cells and detected the expression level of VDR protein and m RNA of HK-2 cells whose VDR gene silenced by Western blot and real-time quantitative PCR. Results Si RNA 1 (6120) could significantly inhibit the expression of VDR in HK-2 cells, and si RNA VDR-1 (6120) stable cell lines were obtained. Conclusion VDR-si RNA 1 (6120) can effectively silence the expression of VDR in HK-2 cells, and we can detect the expression of Na DC1 after VDR expression is inhibited in the next step.
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Key words:
- HK-2 cells /
- Vitamin D receptor /
- Small interfering ribonucleic acid /
- Hypocitraturia
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