人血管细胞中hCREG1基因启动子对血清饥饿发生应答  

Response of CREG1 promoter to serum starvation in human vascular cells

作　　者：韩雅玲[1] 赵昕[1] 闫承慧[1] 康建[1] 张效林[1] 邓捷[1] 徐红梅[1] 刘海伟[1] 

机构地区：[1]沈阳军区总医院全军心血管病研究所心内科,辽宁沈阳110016

出　　处：《中国病理生理杂志》2008年第8期1483-1489,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30570664);辽宁省自然科学基金资助项目(No.2050426)

摘　　要：目的:为揭示E1A激活基因阻遏子(CREG1)在人血管平滑肌细胞(VSMCs)和人脐静脉内皮细胞(HUVECs)中表达的调控机制,构建人CREG1(hCREG1)启动子报告基因载体,探讨CREG1在不同血管细胞中的表达。方法:在对hCREG1进行生物信息学分析的基础上,以人基因组DNA为模板,PCR方法扩增含有hCREG1基因上游-3677bp序列,构建了hCREG15′上游-3677bp、-2310bp和-945bp序列片段,分别将这3个序列克隆到pMD18-T载体上并定向亚克隆到pEGFP-1报告基因载体-pEGFP-hCREG1-P3677、pEGFP-hCREG1-P2310和pEGFP-hCREG1-P945。将重组质粒瞬时转染VSMCs株HITASY和HUVECs,检测绿色荧光蛋白(GFP)的表达。结果:经测序鉴定证实,3个报告基因载体中插入的PCR扩增序列均与hCREG1基因上游DNA序列完全匹配。瞬时转染体外培养的人VSMCs和HUVECs后,经荧光观察和蛋白质印迹(Western blotting)证实,细胞内存在GFP的表达,并且0.5%FBS培养的HITASY细胞中GFP表达较10%FBS培养的细胞中明显增加。HU-VECs中的GFP表达较人VSMCs明显增加。结论:hCREG1基因启动子报告基因载体构建成功,核心启动子存在于5′上游序列的-945bp-0bp区域,为进一步研究hCREG1基因表达提供了条件。AIM ： In order to explore the regulatory mechanisms of the human cellular repressor of E1A - stimulated genes （ hCREG1 ） in vascular smooth muscle cells （VSMCs） - HITASY and in human umbilical vein endothelial cells （HUVECs）, we clone and construct hCREG1 promoter vector to detect its expression in different vascular cells. METHODS： With the results of biological information, the fragments including -3 677 bp, -2 310 bp and -945 bp upstream sequences of hCREG1 were amplified respectively using human genomic DNA template by PCR. The products were inserted into pMD18 - T vector, and then were subcloned into pEGFP - 1 report vector to obtain the pEGFP - hCREG1 -promoter vectors. The pEGFP- hCREG1 - P3677, pEGFP - hCREG1 - P2310 and pEGFP - hCREG1 - P945 vectors were transfeeted into HITASY cells and HUVECs transiently and the expression of green fluorescent protein （GFP） was detected respectively by Western blotting and fluorescent microscope. RESULTS： It was confirmed that all three PCR fragments inserted into vectors were corrected by sequencing analysis. However, the expression of GFP was different significantly in both types of vascular cells. The expression of GFP in HUVECs was higher than that in HITASY cells. Meanwhile, the expression of GFP in HITASY cells with 0.5% FBS was increased obviously compared to that in HITASY cells with 10% FBS. CONCLUSION： The reporter vectors of hCREG1 promoter are constructed successfully in which the core promoter region might be located in -945 bp -0 bp of 5＇ upstream sequences. This study will provide an experimental basis for exploring the regulation of hCREG1 expression.

关 键 词：启动区(遗传学) 阻遏蛋白质类 基因 hCREG1 

分 类 号：R363[医药卫生—病理学]