氯化钴对人eNOS基因启动子SP3替换改构体转录活性的影响

发布时间：2018-11-14 18:03

【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-repSP3和pGL2-eNOS-insSP1两种改造方法在转录活性上没有显著差别。
[Abstract]:Aim: to construct a modified human vascular endothelial cell nitric oxide synthase gene promoter, and to investigate the activity of nitric oxide synthase gene promoter in human umbilical vein endothelial cell line 12 induced by cobalt chloride. Methods: the primers containing SP1 to replace the SP3 mutation sequence were used to construct the pGL2-eNOS-repSP3 vector of the mutant promoter by PCR. The constructed pGL2-eNOS-repSP3 vector and pGL2-eNOS were transfected into HUVEC-12 cells respectively. Using previously constructed SP1 inserted eNOS promoter pGL2-eNOS-insSP1 as positive control, the transcriptional activity of eNOS promoter was detected and compared under different concentrations of cobalt chloride by double luciferase reporter gene technique. Results: eNOS promoter with SP1 replacement SP3 mutation sequence was successfully constructed. The activity of eNOS promoter increased with the increase of cobalt chloride dose in a certain range, but there was no significant difference between the transcriptional activity of eNOS promoter replaced by SP1 and the eNOS promoter inserted by SP1. Conclusion: there is no significant difference in transcriptional activity between pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1.
【作者单位】：暨南大学组织移植与免疫中心;
【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)
【分类号】：R346

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