摘要
目的探讨过氧化体增殖物激活型受体α(PPARα)的特异性激活物亚油酸对HepG2细胞1型纤溶酶原激活物抑制剂(PAI-1)mRNA表达及其活性的影响和在该基因转录调控中的作用机制。方法用不同浓度亚油酸为诱导因素刺激HepG2细胞,采用半定量RT-PCR法检测PAI-1mRNA水平,发色底物法检测PAI-1的活性变化。构建四个含PAI-1启动子序列从-804^+17间不同长度片段驱动的荧光素酶报告基因质粒,体外瞬时转染HepG2细胞,检测荧光素酶的活性。结果与对照组相比,亚油酸组能使HepG2细胞PAI-1mRNA表达及蛋白活性显著升高(P<0.05,P<0.01),且呈一定剂量依赖性;亚油酸诱导可使PAI-1转录活性显著升高(P<0.01);与转染质粒PAI-pGL3-A(-804/+17)相比较,当转染质粒含有PAI-pGL3-B(-636/+17)、PAI-pGL3-C(-449/+17)时,荧光素酶活性显著降低(P<0.01);共转染PPARα表达质粒(PPARα-pSG5)的细胞在亚油酸诱导下PAI-1转录活性显著升高(P<0.01)。结论亚油酸可以增加HepG2细胞PAI-1mRNA表达及其蛋白活性,调节PAI-1的基因转录,PPARα参与亚油酸对PAI-1基因的表达调控;在PAI-1启动子-804^-636、-449^-276区域内存在亚油酸作用的调控PAI-1基因表达的序列。
Objectives To observe the effect of linoleic acid which is the activator of peroxisome proliferator-activated receptors alpha (PPARα) on plasminogen activator inhibitor type-1 (PAI-1) expression in HepG2 cells,and to investigate the mechanisms how the linoleic acid and PPARα influence and regulate PAI-1 gene. Methods HepG2 cells were exposed to linoleic acid at various concentrations, RT-PCR and ELiSA were used to determine the expression of PAI-1 in HepG2 cells. Four luciferase reporter gene plasmids containing different human PAI-1 gene promoter from -804 to + 17 were constructed and transfected into HepG2 cells. The transcriptional activity of PAI-1 was demonstrated by the luciferase activity. Results Compared with the control , linoleic acid could remarkably increase PAI-1 mRNA expression and protein activity in a concentration-dependent manner(P = 0. 038, P〈 0.001 ), and enhance the level of PAI- 1 transcription significantly ( P〈0.01 ). Compared with the PAI-pGL3-A(-804/+ 17) plasmid, when the plasmids PAI-pGL3-B(-636/+ 17) and PAI-pGL3-C(-449/ + 17) were transfected, linoleic acid depressed the luciferase activity remarkably (P〈 0. 01 ) . When co-transfected with PPARα-pSGS, linoleic acid could increase the level of PAI-1 transcription further(P〈0.01). Conclusions Linoleic acid could regulate PAI-1 mRNA expression and gene transcription. PPARa was involved in the regulation of PAI-1 gene expression. The sequences that could regulate the expression of PAI-1 gene induced by linoleic acid may exist in the areas from -804 to -636 and -449 to -276 of PAI-1 promoter.
出处
《中华老年多器官疾病杂志》
2006年第2期124-127,共4页
Chinese Journal of Multiple Organ Diseases in the Elderly
基金
国家自然科学基金资助项目(30470717)