摘要
背景:血管平滑肌的增殖是动脉粥样硬化及支架内再狭窄发生的重要机制,小鼠E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)可以抑制血管平滑肌的增殖,成为心血管领域新的治疗靶点。目的:构建小鼠CREG小分子RNA干扰真核表达载体,下调小鼠细胞中CREG基因的表达。设计、时间及地点:观察性实验,于2007-04/10在解放军沈阳军区总医院心血管研究所完成。材料:小鼠成纤维母细胞系(NIH3T3)由美国新泽西州Robert Wood Johnson医学院病理实验科李少华教授馈赠。方法:应用RNA干扰在线设计工具设计4对可与小鼠CREG基因cDNA结合的短发夹RNA。通过化学合成法合成并克隆至pEN_mH1c载体中,与目的载体pDS_hpEY进行LR重组得到4种表达不同shCREG片段的表达载体pDS_shCREGs。应用LipofectamineTM2000将pDS_shCREGs转染至小鼠NIH3T3细胞,G418筛选获得稳定转染的细胞克隆。主要观察指标:应用反转录-聚合酶链反应和蛋白免疫印迹分别检测CREG的沉默效率。结果:经酶切和DNA测序证实,4种重组pDS_shCREG载体中插入片段的序列正确。反转录-聚合酶链反应和蛋白免疫印迹证实,4种稳定转染细胞中的CREG表达水平均有不同程度的下降,稳定转染pDS_shCREG1的细胞克隆中CREG基因mRNA和蛋白表达分别降低了87%和70%。结论:成功构建小鼠CREG基因真核干扰表达载体,使体外培养NIH3T3细胞中CREG蛋白表达明显降低。
BACKGROUND： Proliferation of vascular smooth muscle is the major mechanism of atherosclerosis and in-stent restenosis. Cellular repressor of E1 A-stimulated genes （CREG） can inhibit the proliferation of vascular smooth muscle, and become the new therapy target in cardiovascular field. OBJECTIVE： To construct the small molecular CREG RNA eukaryotic expression vector, and down-regulate CREG expression in mice cells. DESIGN, TIME AND SETTING： The observation experiment was performed at Cardiovascular Research Institute, General Hospital of Shenyang Military Area Command from April to October, 2007. MATERIALS： Mice fibroblast （NIH3T3） was provided by Professor Shaohua Li from Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, New Jersey, USA. METHODS： Four pairs of short hairpin RNA which could be linked with CREG were designed by on-line RNAi Design. Chemical synthesis method was used to synthesize and clone short hairpin RNA to the pEN_mHlc vector. After LR were recombined with pDS_hpEY, pDS_shCREGs vectors with 4 types of different impressions shCREG fragments were obtained. LipofectamineTM 2000 was used to transfect pDS shCREGs into NIH3T3 cells and select stably transfected cell clones using G418. MAIN OUTCOME MEASURES： Silencing efficiency of CREG was examined by reverse transcription-polymerase chain reaction and protein immunoblotting, respectively. RESULTS： Restriction enzyme analysis and DNA sequencing results confirmed that sequence of inserted fragment in 4 types of recombinant pDS shCREG vector was correct. Reverse transcription-polymerase chain reaction and protein immunoblotting proved that the CREG level in stably transfected cells was declined to different extents, and the RNA and protein expression of clone cells were decreased 87% and 70% respectively in stably transfected pDS_shCREG1. CONCLUSION： Eukaryotic expression vector of CREG gene was successfully constructed, and expression level of CREG protein in NIH3T3 cells decreased significantly in vitro.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第37期7277-7281,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30570644)~~
