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

摘要/Abstract

摘要： 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~-1600bp)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~-1600bp)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。

Abstract: 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.

中图分类号:

R329.
R285.5

张丽华¹, 贾钰华¹, 孙学刚¹, 邢飞跃², 刘志锋², 宋革², 姜勇². 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[J]. 南方医科大学学报, 2004, 24(10): 1113-1116.

ZHANG Li-hua¹, JIA Yu-hua¹, SUN Xue-gang¹, XING Fei-yue², LIU Zhi-feng², SONG Ge², JIANG Yong². Effect of total Panax notoginseng saponins on the activity of human endothelial nitric oxide synthase gene promoter[J]. Journal of Southern Medical University, 2004, 24(10): 1113-1116.

参考文献

[1] 邢飞跃,赵克森,孙学刚,等.人eNOS启动子驱动的红色荧光蛋白报告基因载体构建与表达[J].中华医学杂志,2002,82(16):1093-6Xing FY,Zhao KS,Sun XG,et al.Construction and expression of red fluorescent protein reporter gene vector containing human eNOS promoter[J].Natl Med J Chin,2002,82(16):1093-6.
[2] 刘成海,熊磊,刘平,等.MTT法观察三七总甙对NIH/3T3成纤维细胞毒性作用与增殖的影响[J].中国中西医结合脾胃杂志,1999,7(4):203-5.Liu CH,Xiong L,Liu P,et al.Cytotoxic influence of Panax notoginseng saponins on NIH/3T3 fibroblast and its cell proliferation with MTT colorometric assay [J].Chin J Integr Trad West Med Gastro,1999,7(4):203-5.
[3] Saura M,Zaragoza C,Cao W,et al.Smad2 mediates transforming growth factor-beta induction of endothelial nitric oxide synthase expression[J].Circ Res,2002,91(9):806-13.
[4] Li H,Wallerath T,Forstermann U.Physiological mechanisms regulating the expression of endothelial-type NO synthase [J].Nitric Oxide,2002,7(2):132-47.
[5] Garcia-Montero AC,Vasseur S,Giono LE,et al.Transforming growth factor beta-1 enhances Smad transcriptional activity through activation of p8 gene expression [J].Biochem J,2001,357(Pt 1):249-53.
[6] 郭爱华,龚小卫,阚文宏,等.p38丝裂原激活蛋白激酶对人胚胎肾293细胞一氧化氮合酶基因转录的调控[J].第一军医大学学报,2003,23(3):206-9.Guo AH,Gong XW,Kan WH,et al.Regulation of inducible nitricoxide synthase gene transcriptional by p38 mitogen-activited protein kinase in human embryonic kidney 293 cells[J].J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao,2003,23(3):206-9.
[7] 刘志锋,姜勇.报告基因技术的理论基础及其应用[J].生理科学进展,2002,33(4):361-3.Liu ZF,Jiang Y.The theory basis and application of reporter gene technique[J] Acta Physiol Prog,2002,33(4):361-3
[8] 赵克森,金丽娟.休克的细胞和分子基础[M].北京:科学出版社,2002.1 80-6.
[9] Nakayama M,Yasue H,Yoshimura M,et al.T-786-》C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm [J].Circulation,1999,99 (22):2864-70.
[10] 尹小川,刘建康,周序珑,等.三七总皂甙对培养的猪主动脉内皮细胞释放一氧化氮的影响[J].中国动脉硬化杂志,1996,4(1):20-3Yin XC,Liu JK,Zhou XL,et al.The effect of total Panax notoginseng saponin on releasing nitric oxide from cultured porcine aorta endothelial cells [J].Chin J Arterioscler,1996,4(1 ):20-3.
[11] German Z,Chambliss KL,Pace MC,et al.Molecular basis of cellspecific endothelial nitric-oxide synthase expression in airway epithelium[J].J Biol Chem,2000,275(11):8183-9.
[12] Zhang R,Min W,Sessa WC.Functional analysis of the human endothelial nitric oxide synthase promoter.Spl and GATA factors are necessary for basal transcription in endothelial cells[J].J Biol Chem,1995,270(25):15320-6.
[13] Ziegler T,Silacci P,Harrison VJ,et al.Nitric oxide synthase expression in endothelial cells exposed to mechanical forces [J].Hypertension,1998,32(2):351-5.
[14] 赵钢,张斌,陈建杰,等.抗纤复方对人α1(Ⅰ)前胶原基因启动子活性的影响[J].中国中西医结合杂志,2002,22(9):694-7.Zhao G,Zhang B,Chen JJ,et al.Effect of anti-fibrosis compound on activity of human α1 ( Ⅰ ) precollagen gene promoter[J].Chin J Integr Trad West Med,2002,22(9):694-7.
[15] 陈伟忠,林勇,谢渭芬,等.苦参碱对肝癌细胞端粒酶活性调控及细胞周期的影响[J].第二军医大学学报,2002,23(5):498-500.Chen WZ,Lin Y,Xie WF,et al.Regulation of telomerase activity and cell cycle by matrine in hepatoma cells in vitro[J].J Second Mil Med Univ,2002,23(5):498-500.
[16] 鹿小燕,张文高,周苏宁,等.三七总皂甙治疗心血管疾病的基础与临床研究概况[J].山东中医药大学学报(J Shandong Univ TCM),2000,24(6):468-70.