氯化钴对人eNOS基因启动子SP3替换改构体转录活性的影响  

Effect of Cobaltous Chloride on the transcription activity of modified human eNOS promoter

作　　者：龙宇天[1] 邢飞跃[1] 

机构地区：[1]暨南大学组织移植与免疫中心

出　　处：《细胞与分子免疫学杂志》2010年第8期754-757,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30471635,30971465);广东省自然科学基金资助项目(04010451,5006033);暨南大学“211工程”基金资助项目(2009年)

摘　　要：目的：构建改造的人血管内皮细胞一氧化氮合酶基因启动子，利用氯化钴诱导的人脐静脉血管内皮细胞-12缺氧模型研究其基因启动子活性变化。方法：含SP1替换SP3突变序列的引物通过PCR构建突变启动子pGL2-eNOS-repSP3载体，将已经构建好的pGL2-eNOS—repSP3载体和pGL2-eNOS分别转染HUVEC-12细胞，并以之前构建的SP1插入eNOS启动子改构体pGL2-eNOS—insSP1为阳性对照，通过双荧光素酶报告基因技术检测并比较不同浓度氯化钴刺激下eNOS启动子转录活性的变化。结果：成功构建了含SP1替换SP3突变序列的eNOS启动子改构体。氯化钴刺激后转染细胞，改构体eNOS启动子活性在一定范围内呈现随氯化钴剂量增加而升高的趋势；SP1替换SP3的eNOS启动子转录活性与SP1插入的eNOS启动子之间无显著差异。结论：pGL2-eNOS—rep—SP3和pGL2-eNOS—insSP1两种改造方法在转录活性上没有显著差别。AIM： To construct modified promoter of human endothelial nitric-oxide synthase （eNOS） and investigate transcriptional activity of the eNOS promoter via a hypoxia model of human umbilical vein endothelial cells-12 （HUVEC-12） stimulated by Cobaltous chloride in vitro. METHODS： The pGL2-eNOS-repSP3 vector was constructed by the SP1-replaced SP3 element with using DNA sitedirected mutation of PCR. The cells were transfected with pGL2-eNOS-repSP3 vector and normal pGL2-eNOS-p vector, repsectively. Compared with another modified eNOS promoter（pGL2-eNOS-insSPl） the transcriptional activity of pGL2-eNOS-repSP3 vector was determined using a double luciferase reporter gene system by the stimulation of different concentrations of Cobaltous Chloride. RESULTS： The pGL2-eNOS-repSP3 vector was constructed successfully. After the stimulation of Cobaltous Chloride, the transcriptional activities of both pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1 increased as the concentration of Cobaltous Chloride increased. But there was not significantly statistical difference between them. CONCLUSION： There is no significant difference on transcriptional activities between the two modified vectors, pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1.

关 键 词：一氧化氮合酶 转染 启动子 内皮细胞 氯化钴 

分 类 号：R392.12[医药卫生—免疫学]