三七总皂甙增强人内皮源性一氧化氮合酶基因启动子活性

Effect of total Panax notoginseng saponins on the activity of human endothelial nitric oxide synthase gene promoter

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 study the mechanism of total Panax notoginseng saponin (tPNS) in regulating the transcription activity of human endothelial nitric oxide synthase (heNOS) gene promoter. Methods With gene recombination technique, we subcloned the heNOS gene promoter sequence (from -1 to -1 600 bp) into the BglⅡ/HindⅢ sites of the firefly luciferase reporter gene vector, pGL2-Basic (Promega), to yield the recombinant plasmid designated as peNOS-Luc. With lipofectamine- mediated co-transfection technique, peNOS-Luc, pGL2-Basic and pCMV-β were cotransfected into NIH3T3 cells, which were treated with lipopolysaccharide (LPS), tPNS and transforming growth factor β1 (TGFβ1) respectively. The relative activities (Luc/β-gal) were subsequently determined in the cell lysates to evaluate the effects of these 3 factors on the activity of heNOS gene promoter. Results Double restriction enzyme digestion and sequencing both confirmed that the recombinant plasmid, peNOS-Luc, was constructed correctly, which could be effectively expressed in NIH3T3 cells. Upon LPS stimulation, the luciferase activity was obviously decreased, contrary to the results of tPNS and TGFβ1 treatment, and between the latter two agents, TGFβ1 produced higher transcription activity. Conclusions A firefly luciferase reporter gene vector containing heNOS gene promoter sequence has been constructed correctly. tPNS can up-regulate the activity of heNOS gene promoter in NIH3T3 cells.