携带GFAP启动子的慢病毒载体介导外源基因在肝星状细胞的特异表达

 目的  旨在构建包含由GFAP启动子介导的荧光素酶的慢病毒载体,检测体内外靶向肝星状细胞的外源基因表达的特异性和效率。方法  通过报告基因实验筛选介导效率较高的GFAP启动子,以CMV 启动子作为阳性对照分别介导荧光素酶在小鼠肝星状细胞(JS1)、人肝星状细胞(LX-2)及人肝细胞(L-02)中的表达。通过尾静脉注射将包装的慢病毒注射到四氯化碳诱导的肝纤维化小鼠体内,通过活体成像和Western blot检测慢病毒颗粒在小鼠体内的分布,并通过免疫双荧光染色检测肝星状细胞的特异表达。结果  人GFAP启动子比小鼠GFAP启动子驱动基因表达的效率高,而且在LX-2和JS1细胞中,人GFAP启动子介导的荧光素酶的活性及表达显著高于L-02细胞。活体成像显示小鼠腹部有荧光素酶的表达。GFAP启动子介导的荧光素酶可以与肝星状细胞的特异性生物标记蛋白GFAP、Desmin和α Sma共定位。结论  成功构建包含由GFAP启动子介导的荧光素酶的慢病毒载体,在体外细胞系以及小鼠体内肝中均可以实现荧光素酶在肝星状细胞中特异性表达。

Lentiviral vector containing GFAP promoter directs targeted expression of exogenous genes in hepatic stellate cells

Objective  To construct a lentivirus vector containing a luciferase gene driven by GFAP promoter,and to evaluate the specificity and efficiency of hepatic stellate cells (HSCs) targeted gene expression in vitro and in vivo.Methods  We constructed lentiviral vectors driving the luciferase gene with the human GFAP promoter or CMV promoter,and then transfected these recombinant lentivirus particles into mouse (JS1) and human (LX-2) HSC lines and a human hepatocyte cell line (L-02).We also transferred the viral vectors by intravenous delivery into mice with CCl4-induced liver fibrosis.We profiled the distribution of lentivirus expression in mice using in vivo imaging and Western blot.We then characterized the HSC-specific expression with double staining immunofluorescence.Results  The human GFAP promoter showed higher promoter activity compared with mouse GFAP promoter, and it could drive luciferase expression in HSCs but not in hepatocytes.In vivo imaging showed that lentivirus distributed in mouse epigastrium and hypogastrium between liver and kidney.The luciferase driven by the GFAP promoter in liver co-localized with HSC specific markers including GFAP,Desmin and α Sma.Conclusions  A lentivirus vector containing GFAP promoter was constructed successfully, which can specifically target luciferase expression both in these cell lines and in HSCs of mice liver in vivo.