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目的:采用RNA干扰(RNA interference,RNA i)技术抑制HERG基因的表达,探讨HERG基因编码钾通道对人成神经细胞瘤细胞(SH-SY5Y)存活及增殖的影响。方法:构建发卡状RNA干扰质粒(shRNA-HERG)转染SH-SY5Y细胞,再分别使用半定量RT-PCR和W estern印迹方法观察shRNA-HERG对SH-SY5Y细胞中HERG基因mRNA及其编码蛋白表达的干扰效果。利用SH-SY5Y细胞的生长曲线和集落形成实验,观察转染shRNA-HERG质粒对SH-SY5Y细胞存活及增殖能力的影响。结果:转染shRNA-HERG干扰质粒后,SH-SY5Y细胞中HERG基因的mRNA水平及其编码的钾通道蛋白表达水平显著下降。与对照组相比,shRNA-HERG使SH-SY5Y细胞生长曲线明显下压,细胞倍增时间延长136.8%。集落形成实验结果显示,无论接种细胞数为50,100或200,shRNA-HERG组克隆形成率均显著下降,与shRNA-control组和未干扰组相比,差异有统计学意义(P<0.01)。结论:所构建的shRNA-HERG表达质粒能有效抑制HERG基因编码钾通道蛋白的表达,明显抑制SH-SY5Y细胞的生长和增殖。
OBJECTIVE: To study the effect of HERG gene encoding potassium channels on the survival and proliferation of human neuroblastoma cells (SH-SY5Y) by inhibiting the expression of HERG gene by RNA interference (RNAi) technique. Methods: SH-SY5Y cells were transfected with shRNA-HERG plasmid. The expression of HERG mRNA and protein in SH-SY5Y cells was detected by semi-quantitative RT-PCR and Western blot respectively. Interference effect of expression. The growth curve and colony formation assay of SH-SY5Y cells were used to observe the effect of shRNA-HERG plasmid transfection on the survival and proliferation of SH-SY5Y cells. Results: The mRNA and protein level of HERG gene in SH-SY5Y cells transfected with shRNA-HERG interference plasmid were significantly decreased. Compared with the control group, shRNA-HERG significantly reduced the growth curve of SH-SY5Y cells and prolonged the cell doubling time by 136.8%. The results of colony formation assay showed that the clone formation rate of shRNA-HERG group was significantly decreased regardless of the number of inoculated cells at 50, 100 or 200, which was significantly different from that of shRNA-control and non-interfering groups (P <0.01). CONCLUSION: The constructed shRNA-HERG expression plasmid can effectively inhibit the expression of HERG gene encoding potassium channel protein and significantly inhibit the growth and proliferation of SH-SY5Y cells.