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目的:观察siRNA表达质粒稳定转染靶向沉默HER2/neu基因对SK-OV-3卵巢癌细胞株的影响。方法:针对HER2/neu基因序列构建短发夹状siRNA真核表达载体(pGenesil-1-HER2/neu),转染卵巢癌细胞株SK-OV-3,G418筛选出稳定转染株后,采用RT-PCR和Western Blot法观察HER2/neu基因的沉默效果;CCK-8比色分析检测细胞体外增殖活力;流式细胞仪检测细胞周期。结果:测序证实成功构建HER2/neu的2个短发夹状siRNA真核表达质粒;稳定转染SK-OV-3细胞后,转染了特异性HER-2/neu siRNA的细胞HER-2/neu mRNA及p185蛋白水平均明显低于亲本细胞及转染无义对照序列的细胞;CCK-8比色分析显示HER-2/neu基因沉默的细胞存活分数明显低于HER-2/neu基因高表达的细胞(P=0.000);流式细胞仪细胞周期分析也显示,HER-2/neu基因沉默的细胞处于凋亡状态及非增殖期的比例明显增加(P均<0.01),而处于合成期的比例却显著减少(P≤0.001)。结论:靶向HER2/neu基因的短发夹状siRNA可从转录及翻译水平有效地沉默HER-2/neu基因;HER-2/neu基因沉默的SK-OV-3卵巢癌细胞增殖明显受抑制。
OBJECTIVE: To observe the effect of stable expression of siRNA expression plasmid on SK-OV-3 ovarian cancer cell line with targeted silencing HER2 / neu gene. Methods: The eukaryotic expression vector short hairpin siRNA (pGenesil-1-HER2 / neu) was constructed according to HER2 / neu gene sequence. The transfected ovarian cancer cell line SK-OV-3 and G418 were selected for stable transfection. The silencing effect of HER2 / neu gene was observed by RT-PCR and Western Blot. The viability of HER2 / neu gene was detected by CCK-8 colorimetric assay. Cell cycle was detected by flow cytometry. Results: Two short hairpin siRNA eukaryotic expression plasmids for HER2 / neu were successfully constructed. The HER-2 / neu siRNA transfected SK-OV-3 cells were stably transfected with HER-2 / neu mRNA and p185 protein levels were significantly lower than that of the parental cells and nonsense control transfected cells; CCK-8 colorimetric analysis showed that HER-2 / neu gene silencing cell survival score was significantly lower than HER-2 / neu gene (P = 0.000). The cell cycle analysis by flow cytometry also showed that the proportion of HER-2 / neu gene silenced cells in apoptosis and non-proliferative phase increased significantly (all P <0.01) The proportion of the period was significantly reduced (P≤0.001). Conclusion: Short hairpin siRNA targeting HER2 / neu gene can effectively silence HER-2 / neu gene at transcriptional and translational level; SK-OV-3 ovarian cancer cells with HER-2 / neu gene silencing were significantly inhibited .