乙型肝炎病毒I97L变异核壳蛋白对诱导HepG2细胞凋亡的影响

The Influence of Expression of I97L Mutant Core Proteins of Hepatitis B Virus on the Induction of Apoptosis of HepG2 Cells

目的研究I97L变异核壳蛋白对诱导HepG2细胞凋亡的影响。方法构建EGFP-野毒株核壳蛋白、I97L变异核壳蛋白融合表达载体(pEGFP-WT和pEGFP-L97),酶切和测序鉴定;将pEGFP-WT、pEGFP-L97和对照质粒分别转染HepG2细胞,筛选阳性细胞株;荧光显微镜观察阳性克隆细胞荧光蛋白表达,Western-blot检测核壳蛋白表达;以TNF-α、Act-D诱导HepG2细胞株凋亡,0、16、32和48h采用流式细胞术检测细胞凋亡,32h时采用共聚焦检测细胞凋亡比例。结果成功建立了融合表达蛋白表达载体;荧光显微镜显示各细胞克隆有较好的荧光蛋白表达,Western-blot检测各细胞系核壳蛋白表达没有差异;16、32、48h时,pEGFP-WT、pEGFP-L97表达细胞株凋亡率均明显低于pEGFP-C1细胞株(P<0.05);32h和48h时,pEGFP-L97细胞株凋亡率明显高于pEGFP-WT细胞株(P<0.05);32h时激光共聚焦检测结果与流式细胞术检测结果一致。结论乙型肝炎病毒I97L变异核壳蛋白对诱导HepG2细胞凋亡的影响与野毒株核壳蛋白不同,与野毒株核壳蛋白相比,I97L变异核壳蛋白对凋亡因素可能更敏感。

Objective To investigate the influence of expression of I97L mutant core protein of hepatitis B virus on the induction of apoptosis of HepG2 cells. Method Expression vectors of wild-type core protein and 197L mutant were constructed and subsequently confirmed by double enzyme digestion and sequencing. HepG2 cells were then transfected with pEGFP-WT, pEGFP-L97 or control vector. Positive cell clones were selected by neomycin. Expression of the core proteins was determined under fluorescent microscope and Western-blot. Positively transfeeted cells were then treated with TNF-α and actinomycin D to induce apoptosis. Production of apoptotic cells was evaluated by flow cytometry and the laser confocal microscopy at various times after treatment. Result The expression vectors pEGFP-WT and pEGFP-L97 were established successfully. The amount of the core protein expressed in wild-type cells was similar to the I97L cells. At 16, 32 and 48 hours, the proportion of apoptotic cells in TNF-α and actinomycin D - treated pEGFP-WT or pEGFP-L97 cells was significantly lower than the pEGFP-C1 cells （P〈0.05）. At 32 and 48 hours, the proportion of pEGFP-L97 cells was significantly higher than pEGFP-WT cells （P〈0.05）. Result from the laser confocal microscopic examination was in agreement with flow cytometric analysis. Conclusion Compared with the wild type core protein, the I97L mutant core protein may increase the sensitivity of HepG2 cells to apoptotic stimuli.