亚油酸调控HepG2细胞PAI-1基因表达的机制研究  

MECHANISM OF LINOLEIC ACID ON THE EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE-1 IN HEPG2 CELLS

作　　者：陈静[1] 叶平[2] 刘永学[3] 王成章[1] 兰青 

机构地区：[1]解放军白求恩国际和平医院干二科,河北石家庄050082 [2]解放军总医院南二科,北京100853 [3]军事医学科学院二所,北京100850 [4]解放军装后卫生局,北京100850

出　　处：《中国应用生理学杂志》2008年第2期133-138,共6页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金资助项目(30470717)

摘　　要：目的:探讨亚油酸对HepG2细胞PAI-1基因表达的影响和调控机制。方法:不同浓度亚油酸刺激HepG2细胞,RT-PCR法检测PAI-1 mRNA水平。构建四个含PAI-1启动子序列从-804至+17间系列缺失体片段驱动的荧光素酶报告基因质粒,转染HepG2细胞,初步确定位于PAI-1启动子序列-804至+17之间的Smad3/4结合元件(SBE)具有调控作用,采用重叠PCR法对该元件进行定点突变并构建相应重组质粒,转染HepG2细胞。WesternBlot法检测亚油酸诱导下HepG2细胞中Smad3和Smad4的蛋白表达水平。结果:①50μmol/L及100μmol/L亚油酸可显著增加PAI-1 mRNA的表达,100μmol/L亚油酸可明显上调PAI-1的转录活性。②亚油酸诱导下,PAI-1启动子-734/-731处SBE缺失后,PAI-1荧光素酶活性显著降低。③亚油酸可以显著增加HepG2细胞中Smad3和Smad4的蛋白表达。结论:亚油酸可从转录水平上调HepG2细胞中PAI-1基因的表达,PAI-1启动子上的Smad3/4结合元件即SBE参与亚油酸对HepG2细胞PAI-1基因的转录调控,这一过程涉及到Smads蛋白及其介导的Smad信号转导通路。Aim： To investigate the molecular mechanism underlying the effect of linoleic acid on plasminogen activator inhibitor type-1 （PAI-1） expression in HepG2 cells. Methods： HepG2 cells were exposed to different concentrations of linoleic acid and PAI-1 expression was determined by RT-PCR and colorimetric assay. Luciferase reporter gene plasmids containing four sequentially truncated fragments of the PAI-1 promoter region （-804 to ＋ 17） were constructed, and plasmids carrying constructs of Smad binding element （SBE）-site directed deletions in PAI-1 promoter were also generated using overlap extention PER and transiently transfected into HepG2 cdls, the transcriptional activity of PAI-1 was demonstrated by the luciferase activity. The effect of linoleic acid on SmacL3 and Smad4 protein levels in cultured HepG2 cells was measured by Western blot analysis. Results： ①Linoleic acid remarkably incressed PAI-1 mRNA expression and transcription in varying concentrations.②The level of PAI-1 transcription was gradually decreased in- duced by linoleic acid when transfected the SBE-site directed-deletions plasmids in PAI-1 promoter at -734/-731. ③Protein levels of both Smad3 and 4 in HepG2 cells were increased by linoleic acid. Conclusion： Linoleic acid regulated the expression of PAI-1 from transcriptional level in HepG2 cells and SBE involved in the regulation, and both Smads protein and Smad signaling pathway acted main role in this procession.

关 键 词：亚油酸 纤溶酶原激活物抑制剂-1 HEPG2细胞 SMAD信号通路 

分 类 号：Q786[生物学—分子生物学]