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目的:体外转录合成生存素(survivin)基因siRNA,观察其在骨肉瘤细胞株U-2OS中抑制survivin基因后细胞增殖及细胞凋亡情况。方法:将U-2OS细胞分为A组(空白对照组)、B组(非特异性转染组)、C组(特异性转染组),在荧光显微镜下观察转染前后细胞形态的改变,用RT-PCR和Western blot法分别检测转染前后survivin基因mRNA和蛋白的干涉效果,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡情况,并比较各组结果。结果:荧光显微镜下可见转染后细胞增殖变慢, 凋亡细胞增加;RT-PCR结果显示survivin siRNA明显抑制survivin基因mRNA表达。Western blot法检测C组蛋白阳性表达率比A、B两组显著降低(P<0．01);特异性转染组的细胞增殖缓慢,与其它两组比较有显著性差异(P<0.01)。流式细胞仪检测C组细胞凋亡率与其他两组比较有显著性差异(P<0．01)。结论:体外转录合成特异性survivin siRNA能有效抑制U- 2OS细胞中survivin,in mRNA及蛋白表达,从而抑制细胞增殖,诱导细胞凋亡。RNA干扰技术为骨肉瘤的基因治疗提供了一种新策略。
OBJECTIVE: To transcribe survivin gene siRNA in vitro and observe its effect on the proliferation and apoptosis of survivin gene in osteosarcoma cell line U-2OS. Methods: U-2OS cells were divided into group A (blank control group), group B (nonspecific transfection group) and group C (specific transfection group). The morphological changes of cells were observed under a fluorescence microscope before and after transfection. The interference effect of survivin mRNA and protein before and after transfection was detected by RT-PCR and Western blot respectively. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The results of each group were compared. Results: Fluorescence microscopy showed that after transfection, the proliferation of cells slowed down and the number of apoptotic cells increased. The results of RT-PCR showed that survivin siRNA significantly inhibited the expression of survivin mRNA. Western blot showed that the positive rate of protein expression in group C was significantly lower than that in group A and B (P <0.01). The cell proliferation of group C was slower than that in group A and B (P <0.01) . Flow cytometry C group apoptosis rate compared with the other two groups were significantly different (P <0.01). Conclusion: The transcription of survivin siRNA in vitro can effectively inhibit the expression of survivin, in mRNA and protein in U-2OS cells, thereby inhibiting cell proliferation and inducing apoptosis. RNAi technology provides a new strategy for the gene therapy of osteosarcoma.