转染HBx诱导肝癌细胞株Bel-7404多药耐药的初步研究

摘要

目的 研究HBx转染对肝癌细胞株Bel-7404增殖、细胞周期以及多药耐药的影响,并探讨HBx转染诱导肝癌多药耐药发生的可能机制.方法 选用人肝癌Bel-7404细胞株进行转染,根据HBx转染情况将细胞分为三组,分别为转染HBx细胞组(Bel-7404-HBx组)、转染空载体细胞组(Bel-7404-con组)和空白细胞组(Bel-7404组);实时定量PCR检测各组细胞中HBx RNA的表达.Western blot法检测各组细胞中HBx、Notch-1和多药耐药蛋白(MRP)的表达,CCK-8法检测各组细胞增殖活性和多药耐性,流式分析仪检测各组细胞生长周期.结果 PCR扩增凝胶电泳显示Bel-7404-HBx组有HBx片段出现,而其他两组未见HBx mRNA的表达;Western bolt结果显示在Bel-7404-HBx组处有蛋白条带(即HBx蛋白),而Bel-7404组和Bel-7404-con组未见表达.与Bel-7404组和Bel-7404-con组比较,Bel-7404-HBx组细胞增殖显著加快(P＜0.05);与Bel-7404组和Bel-7404-con组比较,Notch-1和MRP在Bel-7404-HBx组的蛋白表达增加;细胞周期结果显示,与Bel-7404组和Bel-7404-con组比较,Bel-7404-HBx组Go/G1期细胞显著减少(P＜0.05),S期显著增加(P＜0.01),G2/M期变化不大(P＞0.05);多药耐药实验显示,与Bel-7404组和Bel-7404-con组比较,丝裂霉素、阿霉素、5-氟尿嘧啶、顺铂和奥沙利铂在Bel-7404-HBx组中耐药指数明显增加(P＜ 0.05或P＜ 0.01).结论 转染HBx的Bel-7404细胞可能通过诱导Notch-1与MRP的过表达,最终导致肝癌细胞株Bel-7404增殖能力增强和多药耐药的发生.%Objective To study the effects of HBx transfection on proliferation,cell cycle,and multidrug resistance of hepatocellular carcinoma cell line Bel-7404,and to investigate the possible mechanism of HBx transfection in inducing the multidrug resistance of liver cancer.Methods Human hepatocellular carcinoma cell line Bel-7404 was transfected,and the cells were divided into three groups according to the transfection conditions,which were transfected HBx cell group (Bel-7404-HBx group),transfected cell group with empty vector (Bel-7404-con group) and blank cells group (Bel-7404 group).The expression of HBx RNA in all groups was detected by real-time PCR.The expression of HBx,Notch-1 and multi-drug resistance protein (MRP) in all groups was detected by Western blot,the cell proliferation activity and multidrug resistance in all groups were determined by CCK-8,and the cell growth cycle was analyzed by flow cytometer.Results PCR amplified gel electrophoresis showed that HBx fragments were detected in Bel-7404-HBx group,while no HBx mRNA expression could be found in the other two groups;Western blot indicated that the protein band (HBx protein) was detected in the Bel-7404-HBx group,while no expression was found in Bel-7404 group and Bel-7404-con group.Furthermore,compared with Bel-7404 group and Bel-7404-con group,the cell proliferation in Bel-7404-HBx group was increased significantly (P ＜ 0.05);compared with Bel-7404 group and Bel-7404-con group,the protein expression of Notch-1 and MRP in Bel-7404-HBx group was increased significantly;the results of cell cycle showed that,compared with Bel-7404 group and Bel-7404-con group,the number of cells in G0/G1 phase was decreased significantly (P ＜ 0.05),which was significantly increased in S phase (P ＜ 0.01),while there was no significant differences in G2/M phase (P ＞ 0.05);multidrug resistance experiments showed that,compared with Bel7404 group and Bel-7404-con group,the resistance index of Mitomycin,Adriamycin,5-Fluorouracil,Cisplatin and Oxaliplatin in the Bel-7404-HBx group was increased significantly (P ＜ 0.05 or P ＜ 0.01).Conclusion Bel-7404 ceils transfected by HBx may induce overexpression of Notch-1 and MRP,leading to the increase of multiplication capacity of hepatocellular carcinoma cell line Bel-7404 and the occurrence of multidrug resistance.