hi FGF2真核表达载体的构建及其高表达对细胞凋亡的影响  

Construction of Hi FGF2 eukaryotic expression plasmids and its over-expression induced cell apoptosis

作　　者：陈钟琳 姜红岩[2] 洪晓冰[2] 陈中华[2] 郑燕珊[2] 徐涵[2] 石刚刚[2] 黄展勤[2] 

机构地区：[1]汕头大学医学院附属肿瘤医院 [2]汕头大学医学院药理学教研室,广东汕头515041

出　　处：《中国药理学通报》2014年第11期1535-1538,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 30901810);广东省自然科学基金资助项目(No 9151503102000013)

摘　　要：目的构建hi FGF2(high molecular weight isoform fibroblast growth factor-2,hi FGF2)真核表达载体,并观察其过表达后对细胞凋亡的影响。方法设计合成hi FGF2 cDNA模板引物,Nhel和Hind III双酶切pDsRed1-N1质粒,T4DNA连接酶重组hi FGF2质粒,PCR扩增目的基因,琼脂糖凝胶电泳检测及测序鉴定。将重组hi FGF2质粒瞬时转染HEK293细胞,荧光倒置显微镜检测转染效率。AnnexinV-FITC/PI双染法流式细胞仪检测细胞凋亡。结果 hi FGF2真核表达载体符合设计要求,瞬时转染HEK293细胞的转染率达70%以上。过表达hi FGF2,HEK293细胞FITC/PI双染阳性率达(29.12±2.81)%,与正常组、转染空载体组差别有显著性(P<0.01或P<0.05)。结论成功构建hi FGF2真核表达载体,过表达hi FGF2导致细胞凋亡。Aim To construct eukaryotic expressing plasmid of hi FGF2 （ high molecular weight isoform fi-broblast growth factor-2,hi FGF2） gene and to investi-gate its effect on apoptosis after its overexpression in HEK293 cells. Methods The DNA template primer was designed and synthesized. The pDsRed1-N1 plas-mids were digested by the restriction enzymes of Nhel and Hind III. The hi FGF2 was ligated with linearized pDsRed1-N1 by T4 DNA Ligase. The recombinant plasmid was identified by endonuclease digestion and sequenced. The recombinant hi FGF2 plasmid was transient transfected into HEK293 cells by Lipofectami-neTM 2000 Reagent. The transfection efficiency was de-tected by fluorescence inversion microscope. The cell＆amp;nbsp;apoptosis was detected by Annexin V-FITC/PI apopto-sis detection kit with flow cytometry analysis. Results The pDsRed1-N1 eukaryotic expression vector was consistent with the design. The recombinant hi FGF2 plasmid was transfected in HEK293 cells. The trans-fection rate was more than 70%. The FITC/PI dyeing rate in hi-FGF2 over-expression HEK297 cells was a-bout （ 29. 12 ＆#177; 2. 81 ）%. Conclusions pDsRed1-N1 eukaryotic expression vector is successfully constructed and transfected into HEK293 cells. Over-expression of hi FGF2 induces cell apoptosis.

关 键 词：高分子量碱性成纤维细胞生长因子 真核表达载体 转染 细胞凋亡 HEK293细胞 流式细胞术 

分 类 号：R512.62[医药卫生—内科学]