乙肝病毒X蛋白对肝癌细胞多药耐药的影响

Effect of HBX protein on the multidrug resistance of hepatocellular carcinoma

目的观察乙肝病毒(HBV)X蛋白对肝癌多耐药性产生的影响,探讨肝癌多药耐药的形成与乙肝病毒感染的关系。方法应用脂质体介导技术稳定转染pcDNA3/HBX质粒至HepG2人肝癌细胞株,噻唑蓝(MTT)法检测阿霉素及丝裂霉素作用下转染前后细胞的存活率,An- nexin/PI法流式细胞分析术检测转染前后细胞在受到5-氟尿嘧啶(5-Fu)作用后的凋亡指数(AI),逆转录-聚合酶链反应(RT-PCR)和Western blot技术分别检测转染前后HepG2内多药耐药相关基因在mRNA和蛋白水平的表达。结果MTT法显示阿霉素和丝裂霉素作用下,转染了pcDNA3/HBX质粒的HepG2细胞的IC50明显高于对照组(P<0.05)。5-Fu作用后转染组和对照组的AI分别是(9.83±1.50)%VS(17.79±1.70)%,两者差异有统计学意义(P<0.05)。整合了HBX基因的HepG2细胞内多药耐药相关基因在mRNA和蛋白水平的表达也均高于未转染细胞,转染前后比较在mRNA水平mdr1、MRP1、LRP基因的表达分别提高了190%、68%、95%;而在蛋白水平p-gp、MRP1、LRP的表达分别提高了64.3%、87.5%和90.8%。结论乙肝病毒X蛋白可上调HepG2细胞内多药耐药相关基因的表达,从而促进肝癌多药耐药性的产生。

Objective To explore the effects of HBX protein on the formation of multidrug resistance of hepatocellular carcinoma （HCC） and identify the relationship between MDR of HCC and HBV infection. Methods Liposome carrying pcDNA3/HBX plasmid was transfected into HepG2 cells. MTT method was used to determine the viability of the transfected and non-transfected HepG2 cells exposed to ADM or MMC. Apoptosis index （AI） of the transfected HepG2 cells was analyzed by Annexin/PI method using flow cytometry after administration of 5-Fu. The expression of a cohort of the MDR related genes at mRNA and protein levels was detected by RT-PCR and Western blot respectively. Results MTT assay revealed that IC50 of the transfected HepG2 cells exposed to ADM or MMC was significantly higher than that in the control group （ P 〈 0.05）. AI in the transfection group and control group was （9.83 ± 1.5 ） % and （ 17.79 ± 1.7） % respectively after administration of 5-Fu （ P 〈 0.05 ）. The expression of a cohort of the MDR related gene in HepG2 cells transfected with pcDNA3/HBX plasmid was higher than non-transfected cells, that was, at mRNA level, the copies of mdrl, MRP1, LRP gene in transfected cells were respectively higher 190% ,68% ,95% than those in non-transfected cells; at protein level, the amounts of p-gp, MRP1 ,LRP protein in the transfection group were respectively higher 64.3% ,87.5% and 90.8% than those in control group. Conclusion HBX protein could up-regulate the expression of a cohort of the MDR related genes in HepG2 cells and facilitate the development of MDR of HCC.