【摘要】 目的建立乙肝病毒(HBV)HBx基因稳转细胞系,并观察该细胞系的增殖活性及内质网应激相关基因的表达情况。方法采用RT-PCR法从HepG2.2.15细胞株中克隆HBx基因编码区片段,构建pcDNA3.1-HBx真核表达质粒。利用脂质体将pcDNA3.1-HBx转染至人HepG2细胞系,通过G418筛选后,经Western blot法对HBx的表达进行验证。并用MTT法检测细胞的增殖活性,同时观察过表达HBx蛋白对内质网应激相关基因表达的影响。结果成功构建了HBx的真核表达质粒;建立了HBV HBx基因稳转细胞系;MTT法检测显示该细胞系增殖活性明显上升;同时也发现HBx基因的过表达可上调内质网应激相关基因BiP、PERK、ATF6、XBP1、MANF的表达,而CHOP的表达降低。结论 HBx基因过表达可诱导内质网应激,并促进HepG2细胞增殖。更多还原

【Abstract】 Objective To establish the cell line stably expressing hepatitis B virus X protein( HBx) by transfection of HBx to HepG2 cells and to investigate the effect of HBx on ER stress and proliferation. Methods HBx gene encoding fragment cloned from HepG2. 2. 15 cells by RT-PCR was inserted to pcDNA3. 1 vector to construct the pcDNA3. 1-HBx eukaryotic expression plasmid and established the stable cell line after G418 screening. Cell proliferation was determined by MTT method. The expressions of UPR genes were detected by PCR method. Results The HepG2s cell line tably expressing HBx was obtained. Overexpression of HBx up-regulated BiP,PERK,ATF6, XBP1,and MANF and down-regulated CHOP. The cell proliferation was enhanced in the HepG2s cells tably expressing HBx. Conclusion Stably expressing HBx induces ER stress by differentially regulating UPR genes and promotes the cell proliferation.更多还原