中国药理学通报2018，Vol.34Issue(2)：284-289,6.DOI:10.3969/j.issn.1001-1978.2018.02.026
慢病毒介导稳定高表达Cx32的Huh7细胞系建立及其对细胞增殖的影响
Construction of lentiviral vector-mediated Cx32 stably over-expressed Huh7 cell line and its effect on cell proliferation
摘要
Abstract
Aim To construct a lentiviral vector for stable delivery of the connexin32 (Cx32) gene in human hepatocellular carcinoma cell line Huh7,and also to detect its effect on cell proliferation.Methods Human Cx32 gene sequence was obtained by whole gene synthesis and amplified by PCR,and was then inserted into LV5-GFP lentiviral vector to construct the recombinant plasmid LV5-GFP-hCx32.Following identified by restriction endonuclease digestion and DNA sequencing,this plasmid together with lentiviral package plasmid was transfected into 293T cells to produce the lentiviral particles,and the viral titer was assessed under fluorescent microscope.Targeted Huh7 cells were infected with the lentivirus (LV5-hCx32 and LV5-NC),and the infected cells after selection with puromycin were amplified and cultured.The expression and localization of Cx32 were detected by real-time RT-PCR,Western blot and immunofluorescence assay,respectively.The gap junction (GJ) function between adjacent cells was measured by dye transfer assay.The Huh7 cell proliferation capacity was determined by MTT and colony formation assays.Results The results of double enzyme digestion and DNA sequencing proved that the recombinant lentiviral vector LV5-GFP-hCx32 was successfully constructed.After packing in 293T cells,the recombinant lentivirus LV5-GFP-hCx32 with virus drops to 3 × 1011 TU · L-1 was obtained.Huh7 cells transiently infected with the lentivirus LV5-GFP-hCx32 remarkably over-expressed Cx32 at both mRNA and protein levels.Moreover,Cx32 expression was also significantly up-regulated in stably transfected Huh7 cells,and the presence of enhanced functional GJ in intact cells could be detected due to an increased amount of Cx32 protein along the plasma membrane at cell-cell contacts.Compared to LV5-NC group,the proliferation ability in Huh7 cells with recombinant Cx32 declined (P ＜ 0.05).Conclusions The lentiviral vector over-expressing Cx32 gene is successfully constructed,and can stably transfect Huh7 cells to yield sustained over-expression of exogenous Cx32 gene,thus eventually inhibits cell proliferation.关键词
肝细胞癌/缝隙连接蛋白32/慢病毒/载体构建/稳定转染/治疗靶点Key words
hepatocellular carcinoma/connexin32/lentivirus/vector construction/stable transfection/therapeutic target分类
医药卫生引用本文复制引用
纪玉婷,杨燕,郑荣生,张吴琼,刘静..慢病毒介导稳定高表达Cx32的Huh7细胞系建立及其对细胞增殖的影响[J].中国药理学通报,2018,34(2):284-289,6.基金项目
国家自然科学基金资助项目(No 81402514) （No 81402514）
