氯化钴对人的e NOS基因启动子S P1改构体转录活性的影响

摘要

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.%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。