林松挺　陈正义　钟文洲

[摘要] 目的 研究HBx轉染对肝癌细胞株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G0/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。

[关键词] HBx；肝癌细胞株；Bel-7404；Notch信号通路；多药耐药蛋白；多药耐药

[中图分类号] R735.7 [文献标识码] A [文章编号] 1673-7210（2017）09（c）-0016-05

[Abstract] 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 Bel-7404 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 cells 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.endprint

[Key words] HBx； Hepatocellular carcinoma cell line； Bel-7404； Notch signaling pathway； Multi-drug resistance protein； Multidrug resistance

传统意义上的化疗对肝癌的治疗虽然取得了一定的疗效，但是具有较大的毒副作用，其潜在引起了肿瘤细胞的耐药性，并引发肿瘤细胞的扩散与转移[1]。多药耐药是影响化疗疗效以及导致患者死亡的主要原因[2]。肝癌对化疗药物相对不敏感，对其如何发生多药耐药的机制仍不清楚，因此寻找多药耐药的原因尤为关键。HBx由HBV基因组编码，具有多种调控功能，能激活多个与肿瘤侵袭相关的原癌基因及转录因子，是肝细胞癌发病的独立危险因素[3-5]。Notch信号通路是一个重要的高度保守的细胞间信号转导通路，其作用广泛。……