Cai Yuan, He Song, Luo Na, Luo Li, Gong Qian
Department of Gastroenterology, Chongqing Medical University, Chongqing, China.
Zhonghua Gan Zang Bing Za Zhi. 2012 Mar;20(3):211-5. doi: 10.3760/cma.j.issn.1007-3418.2012.03.015.
To investigate the effect of hepatitis B virus (HBV) X protein (HBx) on host cell cycle and HBV replication using cultured primary mouse hepatocytes to gain further insights into the mechanism of HBx-mediated modulation of cell cycle.
Primary cultured mouse hepatocytes were transfected with HBx-expressing (pHBV) or HBx-selected (pHBV triangle X) plasmids, which were generated with sequences of the HBV ayw subtype 1.2 and included the green fluorescent protein (GFP) reporter gene. The levels of cell cycle proteins (p16, cyclin D1, p21, cyclin E and cyclin A) were measured with Western blotting, and HBV DNA was analyzed by Southern blotting and real-time PCR.
The freshly isolated hepatocytes showed no significant differences in levels of cell cycle proteins. However, at 48 hours post-transfection, the levels of cyclin D1, p21 and cyclin E were significantly higher and the level of p16 was significantly lower in the pHBV-transfected hepatocytes than in the pHBV triangle X-transfected hepatocytes (t = 15.713, 22.897, 14.680, and -19.584, respectively, P less than 0.05). The level of cyclin A was not different between the two groups (t = 0.142, P more than 0.05). At 72 hours post-transfection, the level of HBV DNA was higher in pHBV-transfected hepatocytes (rcDNA: 3288.336+/-448.011; dslDNA: 6458.318+/-182.163; ssDNA: 2760.613+/-393.561) than in pHBV triangle X-transfected hepatocytes (rcDNA: 515.721+/-62.530; dslDNA: 2122.228+/-28.347; ssDNA: 1632.013+/-207.021) and in the blank (untransfected) control group (P less than 0.05). Real-time PCR analysis of HBV DNA copy number per cell confirmed these results, (pHBV-transfected: 987.50+/-47.80 vs. pHBV triangle X-transfected: 303.67+/-33.94; t = 20.203, P less than 0.05).
The HBx protein can affect the levels of cell cycle proteins, which may induce quiescent hepatocytes to enter the G1 phase of the cell cycle and stay in this phase instead of entering the S phase, thereby promoting HBV intracellular replication.
利用原代培养的小鼠肝细胞研究乙型肝炎病毒（HBV）X蛋白（HBx）对宿主细胞周期及HBV复制的影响，以进一步深入了解HBx介导的细胞周期调控机制。
用表达HBx的（pHBV）或选择HBx的（pHBVΔX）质粒转染原代培养的小鼠肝细胞，这些质粒由HBV ayw亚型1.2序列构建而成，并包含绿色荧光蛋白（GFP）报告基因。采用蛋白质印迹法检测细胞周期蛋白（p16、细胞周期蛋白D1、p21、细胞周期蛋白E和细胞周期蛋白A）水平，通过Southern印迹法和实时荧光定量PCR分析HBV DNA。
刚分离的肝细胞在细胞周期蛋白水平上无显著差异。然而，转染后48小时，pHBV转染的肝细胞中细胞周期蛋白D1、p21和细胞周期蛋白E水平显著高于pHBVΔX转染的肝细胞，而p16水平显著低于后者（t分别为15.713、22.897、14.680和 -19.584，P均小于0.05）。两组细胞周期蛋白A水平无差异（t = 0.142，P大于0.05）。转染后72小时，pHBV转染的肝细胞中HBV DNA水平高于pHBVΔX转染的肝细胞及空白（未转染）对照组（rcDNA：3288.336±448.011；dslDNA：6458.318±182.163；ssDNA：2760.613±393.561）（P小于0.05）。对每个细胞的HBV DNA拷贝数进行实时荧光定量PCR分析证实了这些结果（pHBV转染组：987.50±47.80 vs. pHBVΔX转染组：303.67±33.94；t = 20.203，P小于0.05）。
HBx蛋白可影响细胞周期蛋白水平，可能诱导静止的肝细胞进入细胞周期的G1期并停滞在此期而非进入S期，从而促进HBV在细胞内的复制。
