强力霉素调控小鼠E1A激活基因阻遏子表达NIH3T3细胞系的建立  

Establishment of a mouse NIH3T3 cells line expressing cellular repressor of E1A-stimulated genes regulated by doxycycline

作　　者：韩雅玲[1] 崔继福[1] 康建[1] 张效林[1] 闫承慧[1] 

机构地区：[1]解放军沈阳军区总医院心内科,辽宁省沈阳市110016

出　　处：《中国组织工程研究与临床康复》2008年第42期8281-8285,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：E1A激活基因阻遏子对血管平滑肌细胞表型的调控;E1A激活基因阻遏子对胚胎早期血管新生的调控:国家自然科学基金资助项目(30070280;30570664);E1A激活基因阻遏子对体外培养人血管平滑肌细胞迁移的影响及其调控的机制:辽宁省科技攻关计划(2007225004-9)~~

摘　　要：背景:前期研究发现,E1A激活基因阻遏子蛋白调控人血管平滑肌细胞的增殖及表型转化,但其表达调控细胞增殖、分化、凋亡的量效关系有待深入探讨。目的:建立强力霉素调控小鼠E1A激活基因阻遏子表达的NIH3T3细胞系,拟为定量观察E1A激活基因阻遏子的生物学功能提供研究基础。设计、时间及地点:对比观察实验,于2006-05/2008-03在沈阳军区总医院全军心血管病研究所实验室完成。材料:强力霉素调控基因表达系统,3T3细胞系。方法:通过反转录-聚合酶链反应方法获得小鼠E1A激活基因阻遏子(mCREG)cDNA序列;将mCREGcDNA序列亚克隆入pRev-TRE载体中,构建强力霉素调控E1A激活基因阻遏子表达pRev-TRE-mCREG载体;分别经G418(新霉素)及潮霉素筛选表达pRev-Tet-On、pRev-TRE-mCREG的NIH3T3细胞克隆;反转录-聚合酶链反应鉴定转染细胞克隆中抗性基因和插入基因的表达。主要观察指标:Westernblotting检测不同浓度的强力霉素诱导后NIH3T3细胞中mCREG的表达。结果:成功构建了强力霉素可调控表达的pRev-TRE-mCREG重组载体;筛选出稳定表达双抗性基因的NIH3T3-mCREG细胞克隆;反转录-聚合酶链反应检测证实细胞克隆中G418及插入基因表达均为阳性;Western blotting检测发现随强力霉素诱导剂量的增加(0,0.01,0.1,1mg/L),NIH3T3-mCREG细胞中mCREG表达呈剂量依赖性增加。结论:成功建立强力霉素调控E1A激活基因阻遏子表达的小鼠NIH3T3细胞系。BACKGROUND： Previous data show that cellular repressor of E1A-stimulated genes （CREG） regulates proliferation and phenotype switch of human vascular smooth muscle cells. Further research is needed to explore the relationships between effects on proliferation, differentiation and apoptosis of cells and dosage of CREG. OBJECTIVE： To establish a mouse NIH3T3 ceils line whose expression of CREG is regulated by the addition of doxycycline, and to provide the basement for the study of biological function of CREG. DESIGN, TIME AND SETTING： A controlled observation was accomplished in Cardiovascular Institute of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA from May 2006 to March 2008. MATERIALS： RevTet-On system and 3T3 cell line were used in this study. METHODS： Mouse CREG （mCREG） cDNA duplicated by reverse transcription-polymerase chain reaction （RT-PCR） from mouse RNA was cloned into a retroviral response vector （pRevTRE） controlled by doxycycline responsive element. The recombinant vector （pRevTRE-mCREG） was constructed and identified by sequence analysis. NIH3T3 cells were transfected by RevTet-On and selected by G418. NIH3T3-RevTet-On clones were transfected by RevTRE-mCREG and selected by hygromycin. The expression of G418 and TRE was detected by RT-PCR analysis in double-stable transfection clones. MAIN OUTCOME MEASURES： The mCREG expression in cell lysate was evaluated by Western blotting after the NIH3T3-mCREG cells cultured in medium of different concentrations of doxycycline. RESULTS： The recombinant pRevTRE-mCREG was constructed successfully. The fragment inserted was confirmed by sequencing The RevTet-On and RevTRE-mCREG was transferred into NIH3T3 cells and the clones （NIH3T3-mCREG） expressed double-resistant genes stably were obtained. RT-PCR showed that NIH3T3-mCREG cells expressed G418 and TRE, respectively. Western blotting analysis demonstrated that the expression of mCREG was upregulated along with the doxycycline increase in a d

关 键 词：阻遏蛋白 腺病毒EIA蛋白 小鼠 

分 类 号：R541[医药卫生—心血管疾病]