乙型肝炎病毒Ⅹ蛋白对原代小鼠肝细胞周期的影响  

Effect of hepatitis B virus X protein on the cell cycle of primary mouse hepatacytes

作　　者：蔡园[1] 何松[1] 罗娜[1] 罗莉[1] 龚倩[1] 

机构地区：[1]重庆医科大学附属第二医院消化内科,400010

出　　处：《中华肝脏病杂志》2012年第3期211-215,共5页Chinese Journal of Hepatology

基　　金：重庆市卫生局医学科研计划项目重点项目（2010-1-37）

摘　　要：目的探讨HBV复制过程中HBx蛋白对原代小鼠肝细胞周期的影响。方法采用原代小鼠肝细胞为研究系统，用野生型HBV质粒（pHBV）和HBx蛋白表达缺失的HBV质粒（pHBV△X）分别转染细胞。Westernblot检测细胞周期蛋白cyclinD1、cyclin E、cyclinA及细胞周期蛋白依赖性激酶抑制因子p16（p16）、p21的表达水平l实时荧光定量PCR和Southernblot检测HBVDNA复制水平。两组之间比较用两样本均数f检验，多样本均数间的比较用方差分析及q检验。结果新分离肝细胞和原代培养24、48、72h肝细胞的细胞周期蛋白表达水平无明显差异；转染48h后，pHBV组细胞与pHBV△X组细胞周期蛋白条带灰度值相比，前者cyclinD1、p21、cyclinE表达量较高，p16的表达量较低，统计量t值分别为15．713，22．897，14．680，-19．584，P值均〈0．05。培养72h后提取HBVDNA，Southernblot检测HBVDNA复制中间体松散环状DNA、双链线性DNA、单链DNA的水平，经条带灰度扫描，pHBV组的水平（分别为3288．336±448．011、6458．318±182．163、2760．613±393．561）明显高于pHBV△X组（分别为515．721±62．530、2122．228±28．347、1632．013±207．021）及空白对照组，P值均〈0．05 实时荧光定量PCR检测HBVDNA复制水平，pHBV组【每个细胞（987．50±47．80）拷贝】也明显高于pHBVAX组【每个细胞（303．67±33．94）拷贝】，t=20．203，P〈0．05。结论HBx蛋白能通过调节细胞周期中各蛋白的表达，使细胞由静止期（G0）进入DNA合成前期（G1）并停滞于G1期，从而促进HBV的复制。Objective 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-modiated modulation of cell cycle. Methods Primary cultured mouse hepatocytes were transfected with HBx-expressing （pHBV） or HBx-delected （pHBV △ X） plasmids, which were generated with sequences of the HBV ayw subtype 1.2 and included the green flourescent protein （GFP） reporter gene. The levels of cell cycle proteins （pl6, 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. Results 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 pl6 was significantly lower in the pHBV-transfected hepatocytes than in the pHBV A X-transfected hepatocytes （t=15.713, 22.897, 14.680, and -19.584, respectively, P 〈 0.05）. The level of cyelin A was notdifferent between the two groups （t = 0.142, P 〉 0.05）. At 72 hours post-transfection, the level of HBV DNA was higher in pI-IBV-transfected hepatocytes （rcDNA： 3288.336 ± 448.011; dslDNA： 6458.318 ± 182.163; ssDNA： 2760.613 ± 393.561） than in pHBV /% 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 〈 0.05）. Real-time PCR analysis of HBV DNA copy number per cell confirmed these results, （pHBV- transfected： 987.50 ± 47.80 vs. pHBV△ X-transfected： 303.67 ± 33.94; t = 20.203, P 〈 0.05）. Conclusions 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 repli

关 键 词：肝炎病毒 乙型 转染 小鼠 肝细胞 细胞周期蛋白质类 HBX蛋白 

分 类 号：R51[医药卫生—内科学] R3[医药卫生—临床医学]