氯化钴对人的eNOS基因启动子SP1改构体转录活性的影响  

Effect of cobaltous chloride on the transcription activity of SP1 modified human eNOS promoter

作　　者：杨云华[1] 樊振华[1] 邢飞跃[1] 

机构地区：[1]暨南大学生命科学技术学院免疫生物学系,广东广州510632

出　　处：《暨南大学学报（自然科学与医学版）》2014年第2期124-129,共6页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然科学基金资助项目(81172824;30971465)

摘　　要：目的:构建改造的人血管内皮细胞一氧化氮合酶(eNOS)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的变化.方法:用不同剂量的氯化钴体外处理细胞复制缺氧模型,PCR点突变技术构建突变启动子pGL2-eNOS-insSP1,分别将pGL2-eNOS-insSP1和pGL2-eNOS-p载体导入HUVEC-12细胞,双荧光素酶报告基因技术用于检测启动子转录活性的变化.结果:DNA测序显示人的eNOS基因启动子SP1改构体的序列正确;改构体eNOS启动子活性在一定范围内随氯化钴刺激剂量的增加而升高.结论:SP1插入改造的方法显著提高了eNOS基因启动子的转录活性.Aim：The study is to construct the modified promoter of eNOS and investigate its function via a hypoxia model induced by cobaltous chloride （CoCl2 ）in primary cultured human umbilical vein en-dothelial cells （HUVEC-12）.Methods：HUVEC-12 cells were cultured with different doses of CoCl2 that is known to mimic hypoxia;the pGL2-eNOS-insSP1 vector was constructed by site-direct mutagene-sis,the cells were transfected with pGL2-eNOS-insSP1 vector and normal pGL2-eNOS-p vector,respec-tively.The transcriptional activity was determined by a double luciferase reporter gene system.Results：The DNA sequencing results demonstrated that the Sp1-modified promoter pGL2-eNOS-insSP1 was suc-cessfully constructed;the transcriptional activity of cells transfected with pGL2-eNOS-insSP1 was signifi-cantly increased by CoCl2 in a certain concentration range.Conclusion：The transcriptional activity of eNOS is significantly elevated by inserting the SP1 binding site into the eNOS promoter.

关 键 词：HUVEC-12 细胞 氯化钴 一氧化氮合成酶 SP1 

分 类 号：R392.11[医药卫生—免疫学] R392.12[医药卫生—基础医学]