B、C基因型乙型肝炎病毒X蛋白调控细胞凋亡的比较研究

The comparative study on the regulation of apoptosis by hepatitis B virus X protein between B and C genotype

目的探讨B、C基因型HBVX蛋白（HBx）对肝癌细胞凋亡的影响方法采用PCR法扩增B、C基因型HBVX基因片段，并定向插入绿色荧光蛋白（GFP）真核表达载体构建重组体pGFP-XB及pGFP-XC；使用FugeneHD将载体pEGFP-C1、pGFP-XB及pGFP-XC转染Bel-402细胞，RT-PCR及WesternBlot鉴定HBVX基因的转录与表达，以建立稳定表达细胞株Bel-402／GFP、Bel-7402／GFP-XB、Bel．7402／GFP-XC，用阿霉素（2．5μg／m1）分别处理上述细胞，处理后用锥虫蓝染色法和流式细胞术检测各组细胞凋亡率。结果RT-PCR及WesternBlot检测显示在Bel-7402／GFP·XB、Bel-7402／GFP-XC有HBVX基因转录与表达。锥虫蓝染色检测表明阿霉素处理的Bel-7402、Bel-7402／GFP细胞发生了明显的时间依赖性死亡，而在Bel-7402／GFP-XB、Bel-7402／GFP-XC和未加阿霉素处理的空白对照组细胞未见明显细胞死亡；流式细胞术检测显示阿霉素处理48h后，Bel-7402／GFP-XB、Bel．7402／GFP-XC细胞的凋亡率分别为3．87％、4．01％，两者差异无统计学意义（P〉0．05），且与未处理的空白对照组细胞凋亡率（2．74％、2．91％、3．00％、2．83％）差异无统计学意义（P〉0．05），但明显低于加入阿霉素处理的Bel-7402细胞（27．05％）及Bel·7402／GFP细胞（29．14％）（P〈0．01）。结论成功建立了稳定表达B、C基因型HBVX与GFP融合蛋白的人肝癌细胞系，为进-步研究HBVX的生物学功能提供了基础。在人肝癌细胞，B、C基因型HBVX蛋白均能抑制阿霉素诱导的细胞凋亡，其抑制细胞凋亡的作用无明显差异。

Objective To investigate the apoptosis regulation on hepatoma cells by HBx between genotype B and C. Methods Genotype B and C HBx gene fragments were amplified and inserted into green fluorescent protein（GFP）eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP-XB and pGFP-XC. The pEGFP-C1 ,pGFP-XB and pGFP-XC were introduced into Bel-7402 cells by Fugene HD to obtain Bel-7402 cells expressing GFP. The transcription and expression of HBx gene were demonstrated by RT-PCR and Western Blot analysis. Bel-7402, Bel-7402/GFP, Bel-7402/GFP-XB, Bel-7402/GFP-XC cells were treated with adriamycin （2. 5 p,g/ml） , and the apoptosis of the cells was determined by trypan blue exclusion,and flow cytometry analysis. Results RT-PCR and Western Blot analysis showed that HBx genes of genotypes B and C were transcribed and expressed in Bel-7402/GFP-XB,Bel-7402/GFP-XC cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in Bel-7402, Bel-7402/GFP cells while no significant cell death was observed in Bel-7402/GFP-XB, Bel-7402/GFP-XC ceils. Flow cytometry analysis indicated that no significant differences of apoptosis rates of Bel-7402/GFP-XB （3.87%） and of Be17402/GFP-XC（4. 01% ） were observed （ P 〉 0. 05 ） , moreover, no significant differences of Bel-7402/ GFP-XB （3.87%）, Be17402/GFP-XC （4. 01% ） and of the untreated cells. Apoptosis rates in Bel-7402/ GFP-XB （3.87%）, Bel-7402/GFP-XC （4.01%）cells were significantly lower than those in Bel-7402 （27.05%） and Bel-7402/GFP （29. 14% ） cells at 48 hours after the adriamycin treatment （P 〈 0. 01 ）. Conclusions Bel-7402 cell lines expressing GFP,GFP-XB and GFP-XC fusion proteins were successfullyestablished. HBV X protein blocks adriamycin-induced apoptosis of Bel-7402 cells. There is no difference between HBx of genotype B and C in inhibiting apoptosis induced by adriamycin.