摘要
目的:获得人早期生长反应1(EGR1)基因,并在人骨肉瘤细胞中瞬时表达,研究该基因在人骨肉瘤细胞中的作用,并探讨其可能的作用机制。方法:以人胎盘组织为模板,巢式PCR(nest PCR)扩增获得EGR1全长基因,克隆入真核表达载体pcDNA3.1(-),酶切、PCR鉴定并测序正确后,命名为pcDNA-EGR1。脂质体法将重组载体转染人骨肉瘤细胞U2OS,实时荧光定量PCR(qPCR)和免疫印迹检测目的基因的表达;MTT法检测细胞生长和增殖情况;流式细胞仪和DAPI染色检测细胞的周期变化和凋亡情况;qPCR检测细胞中与细胞周期、凋亡相关基因的表达变化情况;短发夹RNA(shRNA)干扰EGR1后,检测细胞的变化情况。结果:成功获得了人EGR1基因并构建了其重组表达载体pcDNA-EGR1。EGR1可有效地在U2OS中瞬时表达;可显著抑制细胞增殖(P<0.05),并呈一定的剂量依赖。EGR1可引起细胞周期停滞于G0/G1期,并促进凋亡率增加(P<0.05)。EGR1可引起细胞核出现明显的固缩和凋亡表型。EGR1可引起周期相关基因表达下调,凋亡相关基因表达则显著上调。shRNA干扰EGR1后,细胞凋亡表型和EGR1所调控的相关基因表达水平与对照组一致。结论:EGR1具有抑制骨肉瘤细胞增殖作用,能调节骨肉瘤细胞周期和凋亡相关基因的表达,从而促进细胞周期停滞和凋亡率的增加。
AIM ： To obtain the gene of human early growth response 1 （ EGRI ） and to study its function by transient expression of EGRI in human osteosarcoma cell line U2OS. METHODS： Total RNA was extracted from human placenta and reverse transcription of the RNA to cDNA was performed. The whole length of EGR1 cDNA was isolated by nest PCR and cloned into pcDNA3.1 （ - ）, which was named pcDNA - EGR1. The pcDNA - EGR1 was transfeeted into U2OS cells by LipofectimeTM2000. The over - expression level of EGR1 was detected by real - time quantitative PCR and Western blotting. The effects of EGR1 expression on cell proliferation, cell cycle and apoptosis were analyzed by MTI＇, DA- PI staining and flow cytometry, respectively. RESULTS： The eukaryotic expression vector pcDNA - EGR1 was constructed successfully and high expression level of EGR1 in transfected U2OS cells was observed. Compared with the control cells, the proliferation of U2OS cells transfeeted with EGR1 was significantly inhibited （ P 〈 0. 05）, the cell cycle was significantly arrested at G0/G1 stage and the apoptotie rate was increased （P 〈 0.05 ）. In U20S cells transfeeted with EGR1, over - ex- pression of exogenous EGRI induced nuclear condensation. The expression of CDK2 and CDC2 gene was down - regulated, while the easpase genes were up - regulated（P 〈0. 05）. The apoptosis phenotype and the expression levels of related genes influenced by EGR1 were restored to the levels of control cells by the shRNA method for knocking down of EGR1. CON- CLUSION： EGR1 inhibits the proliferation of U20S cells, arrests the cell cycle and increases the apoptotic rate.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2011年第2期293-299,共7页
Chinese Journal of Pathophysiology
基金
暨南大学211重点学科《生物技术与生物工程药物》第三期预研资助项目
国家自然科学基金重大研究计划项目(No.90919050)
关键词
基因
早期生长反应
瞬时表达
骨肉瘤细胞
生长抑制
Genes, early growth response
Transient expression
Osteosarcoma cells
Growth inhibition
