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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。
OBJECTIVE: To construct a modified promoter of nitric oxide synthase gene in human vascular endothelial cells, and to study the promoter activity of the gene using the cobalt chloride-induced human umbilical vein endothelial cell-12 anoxia model. Methods: Primers with SP1 substitution SP3 mutation sequence were used to construct the mutant promoter pGL2-eNOS-repSP3 by PCR. The constructed pGL2-eNOS-repSP3 vector and pGL2-eNOS were transfected into HUVEC-12 cells respectively. SP1 was inserted into the eNOS promoter pGL2-eNOS-insSP1 as a positive control, and the transcriptional activity of the eNOS promoter under different concentrations of cobalt chloride was detected by dual-luciferase reporter assay. Results: The recombinant eNOS promoter containing SP1 substitution SP3 mutation sequence was successfully constructed. After the cells were transfected with cobalt chloride, the eNOS promoter activity showed a trend of increasing with the dose of cobalt chloride in a certain range. SP1 replaced SP3 eNOS promoter transcriptional activity with the SP1 inserted eNOS promoter No significant difference between. CONCLUSION: There are no significant differences in transcriptional activity between pGL2-eNOS-repSP3 and pGL2-eNOS-insSP1.