一种转导人原代肝细胞的LV-HBx诱导调控表达系统  

An inducible expression system of LV-HBx for transducing primary human hepatocyte

作　　者：周小玲[1,2,3] 熊小芳[2] 谢庆东[1,2,3] 孙平楠[1,2,3] 

机构地区：[1]汕头大学医学院干细胞P2实验室,广东汕头515041 [2]汕头大学医学院生殖医学研究中心,广东汕头515041 [3]广东省感染病与分子免疫病理重点实验室,广东汕头515041

出　　处：《癌变．畸变．突变》2017年第4期266-271,共6页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81570567;81571994);广东省自然科学基金(2014A030313483;2015A030313447);教育部留学回国人员科研启动基金(第49批);汕头大学医学院李嘉诚基金

摘　　要：目的:建立一种在人原代肝细胞中可诱导调控表达HBx的慢病毒表达系统,以便研究HBx生物学特性及其对人肝细胞功能的影响。方法:采用Tet-on系统构建四环素诱导调控的慢病毒-HBx表达系统,并用流式细胞术对其进行优化以达到较高的表达效率。应用该系统制备LV-HBx和LV-rtTA重组慢病毒共同转导肝癌细胞系HepG2和人原代肝细胞后,再加入强力霉素(Dox)诱导调控HBx表达,并采用实时荧光定量PCR(qPCR)检测人原代肝细胞中肿瘤转移相关基因1(MTA1)、血管内皮生长因子A(VEGFA)、转化生长因子β1(TGFB1)、上皮型钙黏附素1(CDH1)、胰岛素样生长因子结合蛋白3(IGFBP3)mRNA的表达水平。结果:重组慢病毒-HBx表达系统构建成功,LV-HBx和LV-rtTA重组慢病毒用量配比经过优化,采用1∶1或2∶1转导后可达到较高的表达效率,Dox诱导产生的表达HBx的阳性细胞率分别为67.3%和66.7%。应用该系统将HBx基因转导入肝癌细胞系HepG2和人原代肝细胞后,可用Dox进行诱导调控HBx的高效表达。其中人原代肝细胞中Dox诱导组的HBx表达水平约为对照组(不加Dox诱导)的45倍,经qPCR检测高表达HBx的人原代肝细胞中MTA1、VEGFA、TGFB1 mRNA的表达水平较对照组均显著升高(P均<0.05),而CDH1、IGFBP3 mRNA表达水平显著降低(P均<0.05)。结论:成功建立了转导人原代肝细胞的LV-HBx四环素诱导调控表达系统,该系统的建立为研究HBx等乙肝病毒基因对人原代肝细胞的影响打下了基础。OBJECTIVE：To construct an inducible lentiviral system which expressed HBx in primary human hepatocytes （PHH） for the study of biological characters of HBx and its influence on PHH.METHODS：We constructed a Tet-on inducible HBx-lentiviral expression system,and optimized it to a relatively high expression level. We applied this system to make LV-HBx and LV-rtTA recombinant lentivirus,to transduce HepG2 hepatoma cells and PHH,and to induce expression of HBx with doxycycline.RESULTS：We successfully constructed the recombinant lentiviral vector of HBx. The ratio of recombinant lentivirus of LV-HBx and LV-rtTA was optimized and a higher transduction efficiency could be approached at the ratio of 1：1 or 2：1 with HBx-positive cell ratio of 67.3% and 66.7%,respectively. This system was successfully applied in transducingHBx gene and inducing HBx expression with doxcycline in HepG2 and PHH. The expression level of HBx in PHH with Dox induction was about 45 fold of the group without Dox induction. HBx significantly increased the expression level of MTA1,VEGFA,and TGFB1 and decreased the expression level of CDH1 and IGFBP3,which is consistent with the previous reports.CONCLUSION：We successfully constructed a Tet-on expression system of LV-HBx for the transduction of HBx into PHH. This system will facilitate studies on influence of HBx and other HBV gene on PHH.

关 键 词：HBX 人原代肝细胞 诱导表达 慢病毒载体 

分 类 号：Q291[生物学—细胞生物学]