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目的探讨RNA干扰(RNAi)抑制卵巢癌细胞表皮生长因子受体-2(HER-2)基因的表达及其细胞效应。方法实验分为以下3组空白对照组(未经转染的人卵巢癌SKOV-3细胞)、转染非特异性的siRNA组及转染特异性的siRNA组。干涉后5d加顺铂。采用半定量PCR技术(RT-PCR)和Western印迹法检测HER-2mRNA和蛋白的表达,用流式细胞仪检测细胞凋亡,用四甲基偶氮唑盐(MTT)比色法检测细胞对顺铂的化学敏感性的变化,分析观察细胞生物学特性的改变。结果HER-2siRNA对HER-2mRNA表达明显抑制,作用能持续10d左右;干涉第7天后开始出现蛋白质表达的明显减弱,转染特异性siRNA组的蛋白质阳性表达率为(25·5±0·8)%,非特异性siRNA组为(95·7±0·8)%,空白对照组为(96·6±1·2)%,前组与后两组比较差异有统计学意义(均P<0·001)。干涉后随着HER-2mRNA表达下降,细胞凋亡增加,第6天凋亡率最高,达(53·2±1·0)%,转染非特异性siRNA组为(4·1±0·3)%,空白对照组为(4·1±0·3)%,差异有统计学意义(P<0·001);干涉后,转染特异性siRNA组的细胞存活率为(58·4±0·8)%,转染非特异性siRNA组为(68·0±0·6)%,空白对照组为(67·0±0·3)%,前组与后两组比较差异有统计学意义(均P<0·001)。结论体外合成的siRNA能有效抑制SKOV-3细胞中HER-2表达,促进细胞的凋亡,提高细胞对顺铂的敏感性,RNAi为卵巢癌的基因治疗提供了一种新策略。
Objective To investigate the effect of RNA interference (RNAi) on the expression of epidermal growth factor receptor-2 (HER-2) gene and its cellular effect in ovarian cancer cells. Methods The experiment was divided into the following three groups of blank control group (untransfected human ovarian cancer SKOV-3 cells), transfected with non-specific siRNA group and transfected specific siRNA group. 5d after intervention plus cisplatin. The expression of HER-2 mRNA and protein was detected by semi-quantitative PCR (RT-PCR) and Western blotting. Apoptosis was detected by flow cytometry. Cell pairs were detected by MTT colorimetric assay Changes in the chemosensitivity of cisplatin were analyzed to observe changes in cellular biological characteristics. Results HER-2 siRNA significantly inhibited the expression of HER-2 mRNA, and the effect lasted for about 10 days. After 7 days of intervention, the expression of HER-2 mRNA was significantly attenuated. The positive rate of protein expression in transfected siRNA group was (25.5 ± 0.8) ),% (95.7 ± 0.8%) in the non-specific siRNA group and (96.6 ± 1.2%) in the blank control group, with significant difference between the former group and the latter two groups (all P < 0 · 001). The apoptosis rate increased with the decrease of HER-2 mRNA expression after intervention, with the highest apoptosis rate (53.2 ± 1.0%) on the 6th day and (4. 1 ± 0.3) )%, And the control group was (4.1 ± 0.3)%, the difference was statistically significant (P <0.001). After interference, the cell viability of transfected siRNA group was (58.4 ± (68.0 ± 0.6)% in the untransfected siRNA group and (67.0 ± 0.3%) in the blank control group. The difference between the former two groups was statistically significant Significance (all P <0.001). Conclusion The siRNA synthesized in vitro can effectively inhibit the expression of HER-2 in SKOV-3 cells, promote the apoptosis of cells and increase the sensitivity of cells to cisplatin. RNAi provides a new strategy for the gene therapy of ovarian cancer.