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利用小鼠白蛋白 (ALB)启动子 /增强子及单纯疱疹病毒胸苷嘧啶激酶 (HSV tk)DNA构建了载体pLLTK ,以研究该载体对肝脏细胞的特异性杀伤效应。首先 ,为了比较载体的肝脏细胞特异转录活性 ,以绿色荧光蛋白 (GFP)基因为报告基因构建了载体 pLE (仅含小鼠ALB启动子 )、pLLE(含小鼠ALB启动子和上游增强子 )和 pLEL(含小鼠ALB启动子和下游增强子 ) ,分别转染到人肝细胞株Hep G2与小鼠乳腺上皮细胞株HC 11,荧光显微镜与流式细胞术分析GFP的表达。然后将载体pLLTK转染到Hep G2研究对细胞的杀伤效应。结果发现 :小鼠ALB启动子 /增强子能驱动GFP肝脏特异表达 ;HSV tk在Hep G2表达使细胞具有更昔洛韦 (GCV)敏感性 ,在GCV作用 7d后 ,MTT分析细胞的生存率 ,pLLTK转染细胞表现明显的细胞死亡 (5 3% ) ,而阴性对照组pcDNA3 1转染细胞没有明显变化 (仅 2 %细胞死亡 )。以上结果表明所有的载体具有肝脏细胞特异性 ,为利用该载体产生肝脏损伤的转基因小鼠提供了细胞水平的实验依据
The vector pLLTK was constructed using mouse albumin (ALB) promoter / enhancer and herpes simplex virus thymidine kinase (HSV tk) DNA to study the specific killing effect of the vector on liver cells. First of all, in order to compare the liver cell-specific transcriptional activity of the vector, the vector pLE (mouse ALB promoter only), pLLE (mouse ALB promoter and upstream enhancer) were constructed using the green fluorescent protein (GFP) And pLEL (containing mouse ALB promoter and downstream enhancer) were transfected into HepG2 cells and mouse mammary epithelial cell line HC11 respectively. The expression of GFP was analyzed by fluorescence microscopy and flow cytometry. The vector pLLTK was then transfected into Hep G2 cells to study the killing effect on the cells. The results showed that the mouse ALB promoter / enhancer could drive GFP liver specific expression. The expression of HSV tk in Hep G2 cells was sensitive to ganciclovir (GCV). After 7 days of GCV treatment, The pLLTK transfected cells showed significant cell death (53%), while the negative control group pcDNA3 1 transfected cells did not change significantly (only 2% cell death). The above results indicate that all vectors have liver cell specificity and provide an experimental basis for cellular levels of transgenic mice that produce liver damage using this vector