HBx基因缺失突变体HBx-d382对L02细胞增殖及非锚定依赖生长能力的影响  

HBx gene deletion mutant (HBx-d382) enhances the proliferation and anchorageindependent growth of L02 cells

作　　者：胡志亮[1] 谭德明[1] 侯周华[1] 谢萍[1] 刘国珍[1] 欧阳奕[1] 刘菲[1] 刘洪波[1] 

机构地区：[1]中南大学附属湘雅医院传染科,湖南省长沙市410087

出　　处：《世界华人消化杂志》2010年第11期1109-1114,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30872228~~

摘　　要：目的:建立稳定表达HBx基因缺失突变体(HBx-d382)的L02肝细胞株,并探讨其对L02细胞增殖的影响.方法:含HBx-d382重组质粒(pcDNA3.0/HBx-d382)经过PCR扩增、双酶切及测序鉴定后,通过脂质体转染和G418筛选获得稳定表达HBx-d382的L02肝细胞株.PCR鉴定基因组中HBx-d382基因整合.RT-PCR和Westernblot鉴定其表达,进一步通过MTT法,软琼脂克隆形成实验检测HBx-d382对L02细胞增殖及非锚定依赖生长能力的影响.用流式细胞仪检测其对细胞周期的影响.结果:经PCR扩增、双酶切及测序鉴定HBx-d382重组质粒构建正确.稳定转染该质粒的L02细胞基因组存在HBx-d382整合.RT-PCR及Westernblot表明在RNA水平和蛋白水平存在HBx-d382表达,稳定表达HBx-d382的L02细胞其增殖能力和非锚定生长能力增强,S+G2期细胞比例升高.结论:成功构建了HBx缺失突变体的真核表达模型,证实HBx缺失突变体能影响细胞增殖,这种效应可能与其影响细胞周期调控相关.AIM： To establish a L02 cell line stably expressing HBx gene deletion mutant （HBx-d382） and to examine their proliferation changes. METHODS： A recombinant plasmid encoding the HBx deletion mutant （pcDNA3.0/HBx-d382） was verified by PCR amplification, double restriction digestion and DNA sequencing, and then introduced into L02 cells by liposome-mediated transfection. Positive clones were selected in the present of G418. The genome integration of the HBx gene deletion mutant was confirmed by PCRamplification, and the expression of the deletion mutant was detected by reverse transcriptionpolymerase chain reaction （RT-PCR） and Western blot. Cell proliferation changes were measured by methyl thiazol tetrazolium （MTT） assay and soft agar colony formation assay. The cell cycle distribution was tested by flow cytometry. RESULTS： The recombinant plasmid pcDNA3.0/ HBx-d382 was verified to contain HBx-d382 by PCR amplification, double digestion and DNA sequencing. Positive clones selected with G418 harbored chromosomally integrated HBx-d382 and could express HBx-d382. This cell line showed enhanced proliferation and anchorage-independent growth as revealed by MTT assay and soft agar colony formation assay. The percentage of cells in S and G2 phases increased in transfected cell line. CONCLUSION： L02 cell line stably expressing the HBx deletion mutant is established successfully. The proliferation ability of this cell line increases probably due to altered cell cycle.

关 键 词：HBX 突变 增殖 细胞周期 

分 类 号：R735.7[医药卫生—肿瘤]