| 陈毅瑶 罗莎 吴涌 李卓 周妙金 邬玲仟 梁德生.内皮细胞特异性启动子pvWF和pVE 的克隆及表达[J].现代生物医学进展英文版,2015,15(17):3201-3206. |
| 内皮细胞特异性启动子pvWF和pVE 的克隆及表达 |
| CloningandExpressionofEndothelialCell-specific Promoter pvWFandpVE |
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| DOI： |
| 中文关键词: 内皮细胞 人胚胎干细胞 特异性启动子 血管性血友病因子 血管内皮钙粘素 基因治疗 |
| 英文关键词: Endothelial cells Human embryonic stemcells Endothelial cell-specific promoter vWF VE-cadherin Gene therapy |
| 基金项目:国家自然科学基金项目（31071301，81000208） |
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| 中文摘要: |
| 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(hESC)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(vWF) 和血管内皮钙粘素
(VE-cadherin/CDH5)。抽提HUVECs的gDNA，通过PCR 扩增内皮细胞特异性表达基因vWF 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pEGFP-N1 中的广谱启动子CMV，构建4 个质粒，即pvWF-1、pvWF-2、pVE-1、
pVE-2，分别转染HUVECs和hESCs，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vWF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hESCs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。 |
| 英文摘要: |
| 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 vWF and VE-cadherin were verified via RT-PCR and immunofluorescence. Promoter fragments of vWF and VE-cadherin gene
were amplified using PCR. The EGFP expression vector originated from pEGFP-N1. The cloned promoters were substituted for the
endogenous CMV promoter. The plasmids: pvWF-1, pvWF-2, pVE-1 and pVE-2 were constructed in total, which were then transfected
into HUVECs and hESCs. 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 hESCs were transfected by the
constructed plasmids, and the GFP expression under fluorescence microscopy showed that pVE-2 promoter was of most specificity and
transcription activity.Conclusion:An endothelial cell specific promoter with the highest GFP specificity and activity in endothelial cells
was successfully cloned. |
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