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目的采用RNA干扰技术逆转人类肝癌细胞系/阿霉素(human hepatocellular liver carcinoma cellline/adriamycin,HepG_2/ADM)细胞中多耐药基因,探讨该基因能否有效地抑制多耐药基因(multid rug resistancegene,MDR1)及其编码的糖蛋白(P-glycoprotein,P-gp)的表达,并检测对HepG_2/ADM耐药表型的逆转效果。方法采用药物大剂量冲击法建立HepG2/ADM耐药模型,构建靶向MDR1的小干扰RNA表达载体,并转染到HepG_2/ADM细胞中,应用半定量逆转录聚合酶链反应检测基因转染前后MDR1 mRNA表达水平的变化,Western blot检测各组细胞P-gp蛋白表达的变化,用噻唑蓝法检测各组细胞对阿霉素、顺铂、长春新碱和氟尿嘧啶等药物的敏感性。结果 HepG_2/ADM细胞系不仅对阿霉素耐药,而且对其他化疗药也有抗性,呈现多药耐药特性;重组质粒鉴定结果表明针对MDR1的小干扰RNA表达载体成功构建;与对照组比较,转入细胞后MDR1mRNA水平明显下降,P-gp蛋白表达明显降低;转入细胞后HepG_2/ADM细胞对化疗药物的敏感性明显提高,部分逆转其耐药性。结论靶向MDR1的小干扰RNA可显著抑制HepG_2/ADM细胞中MDR1基因和P-gp蛋白的表达,逆转P-gp介导的HepG_2/ADM细胞的耐药性。
Objective To reverse the multidrug resistance gene in human hepatocellular carcinoma cell line / adriamycin (HepG_2 / ADM) by RNA interference (RNAi) technology and investigate whether this gene can effectively inhibit multidrug resistance gene , MDR1 and P-glycoprotein (P-glycoprotein, P-gp) in HepG2 / ADM were detected. Methods HepG2 / ADM resistant model was established by high dose shock method. Small interfering RNA expression vector targeting MDR1 was constructed and transfected into HepG2 / ADM cells. Semiquantitative reverse transcription polymerase chain reaction (RT - PCR) MDR1 mRNA expression was detected by Western blot. The expression of P-gp protein in each group was detected by Western blot. The sensitivity of the cells to doxorubicin, cisplatin, vincristine and fluorouracil was tested by thiazolyl blue assay. Results HepG_2 / ADM cell lines were not only resistant to doxorubicin but also resistant to other chemotherapeutic agents, showing multidrug resistance characteristics. The recombinant plasmids showed that siRNA targeting MDR1 was successfully constructed and compared with the control group , The expression of MDR1 mRNA was significantly decreased and the expression of P-gp protein was significantly decreased after transfection into cells; the sensitivity of HepG2 / ADM cells to chemotherapeutic drugs was significantly increased after transfection into cells, partially reversing the drug resistance. Conclusion Small interfering RNA targeting MDR1 can significantly inhibit the expression of MDR1 and P-gp in HepG2 / ADM cells and reverse the drug resistance of P-gp-mediated HepG2 / ADM cells.