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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 ,但不抑制OPNmRNA的转录
Objective: To investigate the effect of osteopontin (OPN) antisense RNA on osteopontin expression in cultured human renal tubular epithelial cells. METHODS: Liposome-mediated transfection of recombinant retroviral vector expressing OPN antisense RNA into rat renal tubular epithelial cell line NRK5 2E and establishment of a renal tubular epithelial cell clone stably expressing OPN antisense RNA Tubular epithelial cell clones expressing OPN cis-sense RNA and blank retroviral vector were used as controls and epidermal growth factor (EGF) was used as a stimulator to detect the above clones by nuclease protection assay (RPA), Western Blot, ELISA and OPN bioassay OPN expression of cells. RESULTS: The OPN antisense RNA was only expressed in antisense clones. The expression of OPN mRNA was detected in antisense clone, cis-sense clone and blank clone cells. EGF increased the expression of OPN mRNA but did not increase the expression of antisense RNA or cis-sense RNA Level; OPN protein expression was not detected in antisense clonal cells with or without EGF and blank clonal cells without EGF, OPN protein expression in cis-sense clonal cells with or without EGF and blank clonal cells plus EGF. CONCLUSION: OPN antisense RNA can inhibit the expression of OPN protein in renal tubular epithelial cells by inhibiting the translation of OPN mRNA but does not inhibit the transcription of OPN mRNA