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目的探讨三七总皂甙(tPNS)对人内皮源性一氧化氮合酶(eNOS)基因启动子转录活性的调控机制。方法以基因重组技术构建人eNOS基因启动子区(-1~ -1 600 bp)驱动的萤火虫荧光素酶报告基因载体peNOS-Luc,采用脂质体介导的细胞基因共转染技术将peNOS-Luc、空载体pGL2-Basic和β-半乳糖苷酶表达质粒pCMV-β共转染NIH3T3细胞,用细菌脂多糖(LPS)、tPNS和转移生长因子β1(TGFβ1)等因子分别刺激转染后的NIH3T3细胞,检测并比较荧光素酶/β-半乳糖苷酶活性,以确定LPS、tPNS和TGFβ1对人eNOS基因启动子转录活性的影响。结果(1)酶切和测序结果均证实重组载体peNOS-Luc构建正确;(2)重组载体peNOS-Luc在NIH3T3细胞中有效表达;(3)在LPS刺激下,人eNOS启动子的转录活性降低,而在tPNS和TGFβ1刺激下,其启动子的转录活性增强,其中,TGFβ1较tPNS所致的转录活性更强。结论正确构建了人eNOS基因启动子区(-1~-1 600 bp)驱动的萤火虫荧光素酶报告基因载体peNOS-Luc;tPNS在NIH3T3细胞中增强人eNOS基因启动子的转录活性。
Objective To investigate the regulatory mechanism of panax notoginseng saponins (tPNS) on the transcriptional activity of human endothelial nitric oxide synthase (eNOS) gene promoter. METHODS: The firefly luciferase reporter gene vector peNOS-Luc driven by the human eNOS gene promoter region (-1 ~ -1 600 bp) was constructed by gene recombination technology. The liposome-mediated co-transfection of the cell genes was used to peNOS- Luc, empty vector pGL2-Basic and β-galactosidase expression plasmid pCMV-β were co-transfected into NIH3T3 cells, and were stimulated with bacterial lipopolysaccharide (LPS), tPNS, and transforming growth factor β1 (TGFβ1), respectively. In NIH3T3 cells, luciferase/β-galactosidase activity was measured and compared to determine the effect of LPS, tPNS and TGFβ1 on the transcriptional activity of the human eNOS gene promoter. Results (1) The results of enzyme digestion and sequencing confirmed that the recombinant plasmid peNOS-Luc was constructed correctly; (2) The recombinant vector peNOS-Luc was efficiently expressed in NIH3T3 cells; (3) The transcriptional activity of the human eNOS promoter was reduced under LPS stimulation. However, under the stimulation of tPNS and TGFβ1, the transcriptional activity of its promoter was enhanced. Among them, TGFβ1 was more transcriptionally active than tPNS. Conclusion The firefly luciferase reporter gene vector peNOS-Luc driven by human eNOS gene promoter (-1~-1 600 bp) was constructed correctly; tPNS enhanced the transcriptional activity of human eNOS gene promoter in NIH3T3 cells.