基因枪转导突变特异性K-rassiRNA对胰腺癌细胞生长的抑制作用

Experimental study of K-ras gene interference and tumor growth inhibition by gene gun transduction of mutation specific K-ras siRNA

目的探讨基因枪转导突变特异性K-ras si RNA对胰腺癌细胞生长的抑制作用。方法根据胰腺癌细胞系突变特征,设计突变特异性K-ras si RNA;应用基因枪将si RNA转导入胰腺癌细胞系,提取总RNA和胞浆蛋白;应用逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Westernblot)方法,检测基因枪转导突变特异性K-ras si RNA对突变型K-ras基因表达的干扰作用;行细胞爬片K-ras p21蛋白免疫组织化学染色,探讨si RNA对K-ras p21蛋白表达的抑制作用;采用CCK-8(cell counting kit-8)活细胞计数法,绘制转基因前后细胞生长曲线,观察si RNA的抑瘤效果。结果RT-PCR结果显示,K-ras突变特异性si RNA,能有效抑制K-ras基因表达(P<0.05);Westernblot和免疫组织化学结果表明,K-ras p21蛋白的表达明显降低(P<0.05);细胞生长曲线显示,转导si RNA组较对照组细胞生长明显受抑制(P<0.05)。结论基因枪可以有效地对小片段双链si RNA进行细胞内转导;突变特异性K-ras si RNA,可显著下调胰腺癌细胞系突变型K-ras mRNA及K-ras p21蛋白表达,并能有效抑制肿瘤细胞的增殖。

Objective To investigate the inhibitory effect of the growth of pancreatic carcinoma cell lines after K-ras siRNA transduction by gene gun. Methods Under the optimum conditions of transduction, pre-designed K-ras siRNA was transduced into pancreatic carcinoma cell lines. After culture for 24 h, RT-PCR and Western blot analysis were carried out to analyze the inhibitory effects of tumor cell growth transducted with K-ras siRNA by gene gun. Immunohistochemistry was performed to detect the expression of K-ras p21 in pancreatic carcinoma cell lines. The cell growth activities were estimated by CCK-8 cell proliferation assay. Results RT-PCR revealed that the cells treated with siRNA had lower transcription of K-ras mRNA than the controls. Western blots and immunohistochemistry indicated a decreased expression, as compared with the controls, of K-ras protein in the cells transducted by siRNA. Mutation specific K-ras antisense oligonucleatides exhibits an anti-proliferate effect in cell proliferation assay. Conclusion K-ras siRNA can significantly down-regulate the transcription and expression of K-ras in mutation positive pancreatic carcinoma cell lines after effective transduction. Transduction of small fragments of double-strand siRNA of K-ras gene has a therapeutic effect in genetic treatment of pancreatic carcinoma.