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目的:探讨RNA干扰诱导型一氧化氮合酶(iNOS)基因的表达对人胆管癌细胞生长的影响。方法:针对iNOS设计并合成3种iNOS-siRNA序列(siRNA1、siRNA2、siRNA3)及阴性对照siRNA序列后,分别转染人胆管癌细胞QBC939细胞,用荧光显微镜下观察转染效率,通过iNOS mRNA与蛋白的变化分析干扰效果,选择干扰效果最为明显的iNOS-siRNA序列,观察其干扰后QBC939细胞增殖、细胞周期及细胞凋亡的变化。实验以无处理的QBC939细胞为空白对照。结果:合成的3种iNOS-siRNA序列均可有效转染人胆管癌QBC939细胞,且转染后均能明显降低QBC939细胞中iNOS mRNA与蛋白的表达(均P<0.05),其中siRNA2对iNOS的抑制作用最为明显,阴性对照siRNA序列对QBC939细胞中iNOS mRNA与蛋白的表达无明显影响(均P>0.05)。转染siRNA2后,QBC939细胞增值明显降低、出现明显的G0/G1期阻滞、凋亡率明显增加(均P<0.05);转染阴性对照siRNA序列的QBC939细胞无上述改变(均P>0.05)。结论:RNA干扰能有效降低iNOS基因在胆管癌细胞中的表达,从而抑制胆管癌细胞的增殖,并促进其凋亡。
Objective: To investigate the effect of the expression of iNOS gene on the growth of human cholangiocarcinoma cells. METHODS: Three iNOS-siRNA sequences (siRNA1, siRNA2, siRNA3) and negative control siRNA sequences were designed and synthesized for iNOS and then transfected into human cholangiocarcinoma cells QBC939 cells. The efficiency of transfection was observed under fluorescence microscope, and the expression of iNOS mRNA was used. The changes of protein were analyzed for interference effects, and the iNOS-siRNA sequence with the most obvious interference effect was selected to observe the changes of proliferation, cell cycle, and apoptosis of QBC939 cells after interference. Experiments were performed on untreated QBC939 cells as blank controls. RESULTS: All three iNOS-siRNA sequences synthesized were successfully transfected into human cholangiocarcinoma QBC939 cells, and the expression of iNOS mRNA and protein in QBC939 cells was significantly reduced after transfection (both P<0.05). Among them, siRNA2 was effective against iNOS. The inhibitory effect was most obvious. The negative control siRNA sequence had no significant effect on the expression of iNOS mRNA and protein in QBC939 cells (all P>0.05). After transfected with siRNA2, the proliferation of QBC939 cells significantly decreased, there was a significant G0/G1 phase arrest, apoptosis rate increased significantly (all P<0.05); the above-mentioned negative control siRNA sequence QBC939 cells did not change (all P>0.05 ). Conclusion: RNA interference can effectively reduce the expression of iNOS gene in cholangiocarcinoma cells, thus inhibiting the proliferation of cholangiocarcinoma cells and promoting their apoptosis.