人肝再生增强因子小干扰RNA表达质粒的构建和鉴定

Construction and identification of expressing siRNA plasmid against human augmenter of liver regenera tion

目的 检测人肝癌细胞株HepG2细胞有无人肝再生增强因子(hALR)的表达,构建针对人肝再生增强因子基因编码区的小干扰RNA(siRNA)表达质粒pSIALR-A及其阴性对照质粒pSIALR-B,观察其对人肝再生增强因子表达的影响。 方法 用免疫细胞化学法观察HepG2细胞表达hALR的情况。将构建成功的pSIALR-A和pSIALR-B分别转染HepG2细胞,转染后48 h,用免疫细胞化学法及逆转录聚合酶链反应法观察人肝再生增强因子蛋白及mRNA的表达。 结果 HepG 2细胞有人肝再生增强因子的表达。成功构建了针对人肝再生增强因子基因编码区的siRNA表达质粒pSIALR-A,并发现它能明显抑制人肝再生增强因子的表达,而随机序列的siRNA却无此作用。 结论 人肝再生增强因子的siRNA具有明显和特异性的抑制人肝再生增强因子的表达作用。

Objectives To detect whether there is an expression of human augmenter of liver regeneration (hALR) in HepG2 cells. To develop a kind of RNAi that specifically targets human augmenter of liver regeneration by synthesizing small interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on hALR expression. Methods The expression of hALR in HepG2 cells was observed with immunocytochemistry. The RNAi plasmid pSIALR-A and the unrelated control plasmid pSIALR-B were transfected into HepG2 cells. Forty-eight hours after transfection, the protein level of hALR was measured with immunocytochemistry; meanwhile, the reverse transcription PCR (RT-PCR) was performed to detect the expression of hALR mRNA. Results hALR was expressed by HepG2 cells. siRNA plasmid pSIALR-A, which targets the cDNA of hALR and the unrelated control plasmid pSIALR-B, was successfully constructed. Both immunocytochemistry and RT-PCR showed that pSIALR-A inhibited the expression of hALR in HepG2 cells significantly, compared with that of pSIALR-B. Conclusion The results showed that the small interfering RNA targeting hALR suppresses the expression of hALR in a sequence-specific manner.