HBx蛋白及其羧基末端缺失35个氨基酸的突变体对正常肝细胞增殖的影响  

Impact of wild-type and carboxylterminal truncated hepatitis B virus X on hepatocyte proliferation

作　　者：朱冉旭 羊东晔 司徒伟基 Ran-Xu Zhu;Dong-Ye Yang;Wai-Kay Seto(Department of Gastroenterology and Hepatology, The University of Hong Kong- Shenzhen Hospital, Shenzhen 518053, Guangdong Province, China)

机构地区：[1]香港大学深圳医院消化及肝病科,广东省深圳市518053

出　　处：《世界华人消化杂志》2018年第13期760-768,共9页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.81702777;广东省自然科学基金资助项目;No.2015A030310053~~

摘　　要：目的构建野生型HBx(wild-type HBx,wt-HBx)和自然突变体HBx(truncated HBx,tHBx~Δ35)重组慢病毒表达载体,建立稳定转染细胞株,观察wt-HBx和tHBx~Δ35对肝细胞增殖和凋亡的影响.方法构建TOPO3.1-wt-HBx和TOPO3.1-tHBx~Δ35重组慢病毒载体;与3个质粒包装系统用磷酸钙方法共同转染到人293T细胞,包装成病毒颗粒,分别感染肝细胞LO2和MIHA,并进行荧光和Western blot法检验细胞中wt-HBx和tHBx~Δ35的表达情况.用细胞计数,细胞活力测定,集落形成实验,流式细胞仪等方法检测对肝细胞增殖和凋亡效应的影响.结果重组慢病毒载体及稳定转染细胞株构建成功,转染后wt-HBx和tHBx~Δ35在细胞中表达明显变化;tHBx~Δ35过表达细胞的增殖速度明显高于wt-HBx组和CTRL对照组,而感染wt-HBx组的肝细胞增殖速度比CTRL对照组和tHBx~Δ35组增殖速度慢(P<0.05);tHBx~Δ35可能通过促进细胞S期的合成而刺激细胞增殖,而wt-HBx可能通过捕获细胞的G0/G1期而抑制细胞增殖.但tHBx~Δ35,wt-HBx在肝细胞的凋亡率是很低的.结论 HBx对肝细胞具有抑制增殖的作用,而tHBx~Δ35对肝细胞具有促进增殖的作用.AIM To construct lentiviral vectors expressing human wild-type HBx （wt-HBx） and truncated HBx （tHBx△35） and study the effect of wt-HBx and tHBx△35 on the proliferation and apoptosis of normal liver cell lines. METHODS Lentiviral vectors TOPO3.1-wt-HBx and TOPO3.1-tHBx△35 were constructed and transfected into 293T cells with three packaging plasmids. The supernatants were collected to infect LO2 and MIHA cells, respectively. The expression of wt-HBx and tHBx△35 was detected by fluorescence microscopy and western blot analysis. The effect of HBx and tHBx△35 on the proliferation, cell cycle, and apoptosis was analyzed by cell counting, MTS, and flow cytometry, respectively. The recombinant lentiviral vectors were successfully constructed. The proliferation of liver cells infected with tHBx△35 was significantly increased, compared with HBx or CTRL infected cells, while the proliferation of liver cells infected with HBx was decreased compared with tHBx△35 or CTRL infected cells （P 〈 0.05）. tHBx△35 improved the number of cells in S phase, while HBx induced G0/G1-S cell cycle arrest. The effect of tHBx△35 or HBx on apoptosis was mild. HBx can inhibit the proliferation of hepatocytes, while tHBx△35 can improve the proliferation of liver cells.

关 键 词：慢病毒载体 tHBx△35 HBX基因 细胞增殖 肝细胞 凋亡 

分 类 号：R373.21[医药卫生—病原生物学]