RNA干扰技术逆转神经胶质瘤细胞多药耐药性  

Reversion of multidrug resistance (MDR) in human glioma cells by RNA interference (RNAi)

作　　者：赵澎[1] 胡微[2] 张亚卓[1] 孙梅珍[1] 何乐[1] 

机构地区：[1]北京市神经外科研究所,100050 [2]北京市垂杨柳医院心内科

出　　处：《中华肿瘤杂志》2006年第3期183-187,共5页Chinese Journal of Oncology

基　　金：北京市科技计划项目(H020920030390)

摘　　要：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 explore whether the constructed vector of short haprin in vivo can induce human glioma cell line BT325 to produce RNAi duplexes and reverse the expression of MDR1 gene. Methods Three 62m oligonucleotide fragments （shRNA） were constructed according to GenBank MDR1 sequence and were cloned to the retrovirus-delivered vectors. After transfected these vectors directly into the human malignant glioma BT325 cells by lipofectamine 2000 with enhanced green fluorescence protein （EGFP） co-transfecting,the MDR1 gene silence effects were detected by the changing level of mRNA and P-glycopretein including real time PCR （RT-PCR）, Northern blot and Western blot analysis. To assess the multidrug resistance against adriamycin （ADR） and VCR, cell proliferation assays were performed by cell counting kit-8. Results The RNAi plasmid vectors were constructed successfully. RT-PCR showed MDR1 mRNA was significantly reduced （P 〈 0.05 ）. Northern blot analysis showed that the gene silence became most intense at 48 hours after transfection. Western blot analysis demonstrated that P-gp expression was reduced at different time to 12. 9%, 30.3% and 4.8% , respectively. The chemosensitivity assays indicated that the transfected cells showed an enhanced sensitivity to ADR and VCR. Based on the value of IC50, BT325 cells had significantly increased sensitivity to the drugs. Conclusion The sequence specific RNAi can inhibit MDR1 mRNA and P-gp expression in the glioma cell line. It may reverse multidrug resistance phenotype, therefore, may provide promising therapeutic modalities in the treatment of human glioma.

关 键 词：神经胶质瘤 RNA干扰 多药耐药性 基因治疗 

分 类 号：R739.4[医药卫生—肿瘤]