内皮细胞特异性启动子pvWF和pVE的克隆及表达

Cloning and Expression of Endothelial Cell-specific Promoter pvWF and pVE

目的:克隆并验证内皮细胞(Endothelial Cells,ECs)特异性启动子,为转染人胚胎干细胞(h ESC)后实时监测ECs的定向分化情况以及利用干细胞实施血友病A的基因治疗研究提供基础。方法:通过酶消化法原代分离人脐静脉内皮细胞(HUVECs),结合RT-PCR和免疫荧光验证分离后的HUVECs表达内皮细胞特异性标志基因血管性血友病因子(v WF)和血管内皮钙粘素(VE-cadherin/CDH5)。抽提HUVECs的g DNA,通过PCR扩增内皮细胞特异性表达基因v WF和VE-cadherin转录起始位点上游不同大小的启动子片段,将其取代报告基因载体p EGFP-N1中的广谱启动子CMV,构建4个质粒,即pv WF-1、pv WF-2、p VE-1、p VE-2,分别转染HUVECs和h ESCs,48 h后观察并比较各启动子片段启动绿色荧光蛋白GFP表达情况,筛选最具特异性及转录活性的启动子片段。结果:通过酶消化法,本研究成功分离出具有典型上皮样细胞的HUVECs。RT-PCR和免疫荧光结果表明HUVECs特异性表达v WF和VE-cadherin。酶切及测序证实所构建的4个含ECs特异性启动子片段的质粒与理论序列相符,通过核转染至HUVECs及h ESCs后,48 h后观察到所克隆的VE-cadherin 2105bp启动子片段具有内皮细胞表达的特异性和较强的转录活性。结论:本研究成功筛选出具有内皮细胞表达特异性及较强转录活性的启动子片段。

Objective： To lay the foundation for hemophilia A gene therapy and real-time monitoring of human embryonic stem cells differentiation to endothelial cells, endothelial cell-specific promoters were cloned and its specificity was validated. Methods： A stable method of obtaining endothelial cells from umbilical vein was established. Then the expression of endothelial cell specific marker gene v WF and VE-cadherin were verified via RT-PCR and immunofluorescence. Promoter fragments of v WF and VE-cadherin gene were amplified using PCR. The EGFP expression vector originated from p EGFP-N1. The cloned promoters were substituted for the endogenous CMV promoter. The plasmids： pv WF-1, pv WF-2, p VE-1 and p VE-2 were constructed in total, which were then transfected into HUVECs and h ESCs. In order to find the strongest and most specific promoter, the expression of GFP 48 h after transfection were observed and compared. Results： Endothelial cells were isolated from umbilical vein and the markers of their specific genes were detectable. Four plasmids were confirmed by restriction enzyme digestion and sequencing. HUVECs and h ESCs were transfected by the constructed plasmids, and the GFP expression under fluorescence microscopy showed that p VE-2 promoter was of most specificity and transcription activity. Conclusions： An endothelial cell specific promoter with the highest GFP specificity and activity in endothelial cells was successfully cloned.