摘要
目的探讨乙型肝炎病毒HBx蛋白对阿霉素诱导的肝癌细胞凋亡的影响。方法将adr亚型HBx基因片段定向插入绿色荧光蛋白（GFP）真核表达载体pEGFPCl，构建重组体pGFP—HBx。将pEGFPCl、pGFPHBx转染HepG2细胞，采用G418筛选抗性克隆、荧光显微镜观察及RT—PCR检测HBx基因表达情况以建立稳定表达细胞株HepG2／GFP、HepG2／GFPHBx。用阿霉素（2．5μg／m1）分别处理HepG2、HepG2／GFP、HepG2／GFPHBx细胞，处理后不同时间在显微镜下观察细胞形态变化，并用锥虫蓝染色计数死亡细胞；流式细胞仪检测阿霉素处理36h后细胞凋亡率。结果HepG2／GFP、HepG2／GFPHBx细胞传70代后，仍表达强的GFP；RTPCR检测显示在HepG2／GFP—HBx细胞有HBx基因转录表达。锥虫蓝染色检测表明阿霉素处理的HepG2、HepG2／GFP细胞发生了明显的时间依赖性细胞死亡，而在HepG2／GFPHBx和对照组细胞未见明显细胞死亡；流式细胞仪检测显示阿霉素处理36h后，HepG2／GFP—HBx细胞凋亡率为3．94％，明显低于HepG2（59．03％）、HepG2／GFP细胞（61．38％）（P〈0．01），而与未处理对照组细胞凋亡率（2．12％，2．78％，2．55％）差异无统计学意义（P〉0．05）。结论成功建立了稳定表达GFP、GFPHBx的HepG2细胞株；HBx能够抑制阿霉素诱导的HepG2细胞凋亡。
Objective To investigate the effect of hepatitis B virus X protein （HBx） on adriamycininduced apoptosis of hepatocellular carcinoma cells. Methods HBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein （GFP） eukaryotic expression vector pEGFP-Cl to construct recombinant pGFP/HBx. The pEGFP-CI and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT- PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin （2.5 μg/ml）, and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis. Results Under a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx （3.94%） cells were significantly lower than those in HepG2 （59.03%） and HepG2/ GFP cells （61.38%） at 36 hours after the adriamycin treatment （P 〈 0.01）. No significant differences of apoptosis rates of HepG2/GFP-HBx （3.94%） and of the untreated cells （2.12%, 2.78%, 2.55%） （P 〉 0.05） were observed. Conclusions A HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2008年第1期25-28,共4页
Chinese Journal of Hepatology
基金
国家自然科学基金（30471522）}教育部留学回国人员科研启动基金
广东省自然科学基金（04009386）
关键词
癌
肝细胞
细胞凋亡
HBX蛋白
肝炎病毒
乙型
阿霉素
绿色荧光蛋白
Carcinoma, hepatocellular
Apoptosis
HBx protein, hepatitis B virus
Adriamycin
Green fluorescent protein
