摘要
本研究建立TF启动子转录活性的荧光素酶基因稳定细胞株,应用该细胞模型筛选调控TF基因表达的药物,并为深入研究其分子机制打下基础。构建TF启动子的一系列5′端截短型的荧光素酶报告基因质粒(包括-2174 bp^+128 bp,-684 bp^+128 bp,-247 bp^+128 bp,-201 bp^+128 bp),将质粒电转染至U937细胞中,建立表达荧光素酶报告基因的稳定细胞株。应用ATRA验证该细胞株的功能;应用bortezomib、尿多酸肽(CDA-II)等药物处理该细胞株24小时,分析荧光素酶基因活性,筛选出能够调控TF基因表达的药物。结果发现,5 nmol/L bortezomib能激活其转录活性,上调TF转录本表达水平;1 mg/ml CDA-Ⅱ抑制TF启动子的转录活性,下调TF转录本的表达水平。TF启动子逐步截短功能分析发现,bortezomib及CDA-ⅡII调控TF启动子转录活性的区域位于-201 bp—0 bp之间。结论:本研究建立了表达TF启动子荧光素酶活性的U937稳定细胞株,并筛选出能够调控TF基因转录的药物CDA-II及bortezomib,为将来筛选新药物及深入研究其分子机制打下了基础。
This study was purposed to screen the drugs for regulating tissue factor（TF） gene expression through establishing stable cell line with luciferase gene having TF promoter transcription activity,so as to provide the basis for further studying the molecular mechanism of screened drugs.A series of luciferase reporter gene plasmids under control of 5′-truncated TF promoter（including-2174 bp —＋128 bp,-684 bp —＋128 bp,-247 bp —＋128 bp and-201 bp —＋128 bp） were constructed.The above plasmids were separately electroporated into U937 cells to establish stably transfected sublines.The function of stable cell line was testified by treatment with ATRA,the luciferase gene activity was analyzed by treating established cell line with bortezomib（BTZ） and CDA-II,and drugs for regulating TF gene expression were screened.The results indicated that the BTZ of 5 nmol/L could activate TF gene transcription activity,up-regulate the expression level of TF transcripts;CDA-II of 1 mg/ml could suppress TF gene transcription activity,down-regulate the expression level of TF transcripts.The functional analysis of TF promoter transcription revealed that the region of regulating TF promoter transcription activity by BTZ and CDA-II was between-201 to 0 bp.It is concluded that stable cell line U937 expressing luciferase activity of TF promoters is established,the novel drugs regulating TF gene expression are screened out by means of this established cell line.This study provides basis for screening the new drugs and further studying thier molecular mechanisms.
出处
《中国实验血液学杂志》
CAS
CSCD
2011年第1期207-210,共4页
Journal of Experimental Hematology
基金
大连医科大学附属第二医院青年基金项目(编号2007)
辽宁省卫生厅医学高峰建设工程项目基金(编号200912)
作者简介
通讯作者：闫金松，主任医师、博士．电话：（0411）84671291-3133．E-mail：yanjsdmu@gmail．com
