CAJ | 학술논문

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。
목적：구건개조적인혈관내피세포일양화담합매（eNOS）기인계동자，이용록화고유도적인제정맥혈관내피세포-12결양모형연구기기인계동자활성적변화。방법：용불동제량적록화고체외처리세포복제결양모형， PCR점돌변기술구건돌변계동자pGL2-eNOS-insSP1，분별장pGL2-eNOS-insSP1화pGL2-eNOS-p재체도입HUVEC-12세포，쌍형광소매보고기인기술용우검측계동자전록활성적변화。결과：DNA측서현시인적eNOS 기인계동자SP1개구체적서렬정학；개구체eNOS계동자활성재일정범위내수록화고자격제량적증가이승고。결론：SP1삽입개조적방법현저제고료eNOS기인계동자적전록활성。
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.