转染HBx诱导肝癌细胞株Bel-7404多药耐药的初步研究  

Primary study on HBx transfection in inducing the multidrug resistance of hepatocellular carcinoma cell line Bel-7404

作　　者：林松挺[1] 陈正义[1] 钟文洲[1] 

机构地区：[1]海南省海口市人民医院消化内科,海南海口570208

出　　处：《中国医药导报》2017年第27期16-20,共5页China Medical Herald

基　　金：海南省自然科学基金资助项目(20158335)

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 m RNA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G_0/G_1期细胞显著减少(P<0.05),S期显著增加(P<0.01),G_2/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 muhidrug 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 muhidmg 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, Ad

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 

分 类 号：R735.7[医药卫生—肿瘤]