植物雌激素α-玉米赤霉醇对HUVEC组织因子基因表达的转录调控作用

Transcription regulation on tissue factor gene expression in the HUVEC by phytoestrogen α-zearalanol

目的探讨植物雌激素α玉米赤霉醇(ZAL)影响血管内皮细胞组织因子(TF)基因表达的转录调控机制,并与内源性动物类雌激素17β联雌二醇(17β联estradiol,E2)进行比较分析。方法进行人脐静脉内皮细胞(HUVECs)原代培养,利用基因重组技术构建4个含人TF基因5′上游不同长度序列的荧光素酶报告基因质粒,用脂质体转染法分别转入以下6组细胞:①正常对照组;②10-7molLE2刺激组;③10-7molLZAL刺激组;④10-6molLAngⅡ刺激组;⑤10-7molLE2+10-6molLAngⅡ刺激组;⑥10-7molLZAL+10-6molLAngⅡ刺激组;测定细胞裂解液中荧光素酶比活性(relativeluciferaseactivity,RLA)的变化。结果转染pGL3TF171+71的各组细胞RLA均较低,不同刺激组之间无显著差异;AngⅡ可使质粒pGL3TF593+71,pGL3TF316+71,pGL3TF236+71荧光素酶表达大量增加,E2、ZAL均可不同程度地抑制AngⅡ的上述效应,2者间无显著差异。结论含有1个NFκB位点和2个AP1位点的-236bp～-171bp区域在E2或ZAL影响TF基因转录中有重要作用。

Objective To investigate the effects of phytoestrogen α-zearalanol (ZAL)on transcription regulation of tissue factor (TF)gene and to compare with endogenous estrogen 17β-estradiol(E_ 2) in cultured human umbilical vein endothelial cells (HUVECs). Methods Using gene recombination techniques, four luciferase reporter gene plasmids containing different length of human TF gene promoter were constructed. With lipofectamine transfection techniques,the above plasmids and pSV-β-Gal as a intracontrol were co-transfected into HUVECs of different groups to study the role of the cis-cting elements on TF gene transcription:1) control;2) 10~ -7mol/L E_ 2;3) 10~ -7mol/L ZAL;4)10~ -6mol/L AngⅡ;5)10~ -7mol/L E_ 2 +0~ -6mol/L AngⅡ;6)10~ -7mol/L ZAL_ +10~ -6mol/L AngⅡ. Results 1)the relative luciferase activity(RLA)of HUVECs transfected with pGL3-TF-171/+71 were very low and there was no significant difference among the six groups;2) AngⅡ could induce pGL3-TF-593/+71, pGL3-TF-316/+71 and pGL3-TF-236/+71 to express luciferase highly,which were inhibited by ZAL and E_ 2. Conclusions The sequence from -236bp to -171 bp containing one NF-κB element and two AP-1 elements was critical in TF gene transcription induced by E_ 2 and ZAL.