反义RNA对培养的肾小管上皮细胞骨调素表达的调节

Regulation of osteopontin expression by its antisense RNA in renal epithelial cells

目的 :探讨骨调素 (OPN)反义RNA对培养的肾小管上皮细胞骨调素表达的影响。方法 :脂质体介导将表达OPN反义RNA的逆转录病毒重组载体转染大鼠肾小管上皮细胞系NRK5 2E细胞 ,建立稳定表达OPN反义RNA的肾小管上皮细胞克隆 ,以转染了表达OPN顺义RNA和空白逆转录病毒载体的肾小管上皮细胞克隆为对照 ,以表皮生长因子 (EGF)为刺激剂 ,通过核酸酶保护分析 (RPA) ,WesternBlot,ELISA和OPN生物活性分析检测上述克隆细胞的OPN表达。结果 :OPN反义RNA仅被反义克隆细胞表达 ,反义克隆、顺义克隆和空白克隆细胞均有OPNmRNA的表达 ,EGF能增加它们OPNmRNA的表达水平 ,但不增加反义RNA或顺义RNA的表达水平 ;加或不加EGF的反义克隆细胞和不加EGF的空白克隆细胞无OPN蛋白的表达 ,加或不加EGF的顺义克隆细胞和加EGF的空白克隆细胞有OPN蛋白的表达。结论 :OPN反义RNA能抑制肾小管上皮细胞OPNmRNA的翻译而抑制OPN蛋白的表达 。

AIM: To investigate the effect of antisense RNA on osteopontin (OPN) expression in renal tubular epithelial cells. METHODS: Cell clone expressing stably OPN antisense RNA was formed by transfering retroviral vector expressing OPN antisense RNA into renal tubular epithelial cells, NRK52E cells, using liposome, with cell clones transfected by empty vector and vector expressing OPN sense RNA as controls. Ribonuclease protection assay(RPA), Western Blot, ELISA and assay of OPN activity were performed to detect expression of OPN mRNA and protein in above clones cultured with or without epidermal growth factor(EGF). RESULTS: The antisense RNA was only expressed by antisense clone. Antisense clone, sense clone and empty clone all expressed OPN mRNA. EGF enhanced expression of OPN mRNA, but not OPN antisense RNA or OPN sense RNA in above clones. OPN protein was not expressed in antisense clone cultured with or without EGF and empty clone cultured without EGF, but was expressed in sense clone cultured with or without EGF and empty clone cultured with EGF. CONCLUSION: Antisense RNA can inhibit OPN protein expression by means of preventing OPN mRNA translation, but not inhibit OPN mRNA transcription in renal tubular epithelial cells.