论文部分内容阅读
目的探讨利用RNA干扰(RNAi)技术逆转神经胶质瘤细胞多药耐药性。方法根据多药耐药基因1(MDR1)的碱基序列设计并合成短发夹RNA(shRNA),构建逆转录病毒质粒载体,用阳离子脂质体法体外转染BT325细胞株,以增强型绿色荧光蛋白(EGFP)表达作为对照。采用定量PCR、Northernblot检测转染前后MDR1mRNA的表达,Westernblot检测蛋白表达;使用CCK8试剂盒对转染后的细胞进行化疗药物敏感性试验,评价RNAi对多药耐药性的逆转作用。结果成功构建RNAi质粒载体。共转染实验组RT PCR定量MDR1mRNA相对表达水平均有所下降(P<0.05);Northernblot表明,转染48h细胞干扰最强;Westernblot显示,siRNA各转染组P糖蛋白(P gp)的表达分别降低12.9%、30.3%和4.8%,在48h抑制最强;而药物敏感试验显示,转染siRNA后细胞对药物的敏感性明显增强。结论RNAi能够明显抑制神经胶质瘤细胞系MDR1mRNA和P gp蛋白的表达,进而对多药耐药性发挥明显的逆转作用,为基因治疗提供了一种新的手段。
Objective To investigate the reversal of multidrug resistance in glioma cells by RNA interference (RNAi) technique. Methods According to the base sequence of multidrug resistance gene 1 (MDR1), a short hairpin RNA (shRNA) was designed and synthesized, and a retroviral plasmid vector was constructed. BT325 cells were transfected by cationic liposomes in vitro. The enhanced green Fluorescent protein (EGFP) expression was used as a control. The expression of MDR1 mRNA was detected by Northern blot before and after transfection. The expression of MDR1 mRNA was detected by Western blot. CCK8 kit was used to test the chemosensitivity of transfected cells to evaluate the reversal effects of RNAi on multidrug resistance. Results RNAi plasmid vector was successfully constructed. The expression of MDR1mRNA in co-transfection group was decreased by RT-PCR (P <0.05). Northern blot showed that the interference was the strongest at 48h after transfection; Western blot showed that the expression of P-gp Decreased by 12.9%, 30.3% and 4.8% respectively, and showed the strongest inhibitory effect at 48h. However, drug sensitivity test showed that the sensitivity of the cells to the drug was significantly enhanced after siRNA transfection. Conclusion RNAi can significantly inhibit the expression of MDR1 mRNA and P gp protein in glioma cell lines, which can reverse the multidrug resistance and provide a new method for gene therapy.