GDNF基因修饰大鼠胚胎中脑神经干细胞的建立  

Establishment of GDNF gene modified midbrain-derived neural stem cells

作　　者：邓兴力[1] 刘如恩[2] 郭京[2] 冯忠堂[1] 王武[3] 雷德强[4] 李红艳[5] 陈志华[5] 

机构地区：[1]昆明医学院第一附属医院神经外科,云南昆明650032 [2]中日友好医院神经外科,北京100029 [3]中日友好医院放射诊断科,北京100029 [4]华中科技大学同济医学院附属协和医院神经外科,湖北武汉430022 [5]中日友好医院临床医学研究所,北京100029

出　　处：《中风与神经疾病杂志》2008年第3期260-264,共5页Journal of Apoplexy and Nervous Diseases

基　　金：国家自然科学基金资助项目(30672151)

摘　　要：目的建立脑源性神经营养因子(GDNF)基因修饰大鼠胚胎中脑神经干细胞。方法以RT-PCR扩增GDNF基因编码序列,将其克隆至质粒pEGFPN1构建重组质粒pEGFPN1-GDNF,经酶切鉴定及序列分析后,以FuGENE HD转染试剂介导转染大鼠胚胎中脑神经干细胞。免疫细胞化学、western blot鉴定GDNF的表达,诱导分化后免疫细胞化学鉴定其分化能力。结果RT-PCR产物为650bp的特异片段,重组质粒pEGFPN1-GDNF经酶切产生650bp和4.7kb的片段,序列分析结果与文献报道一致。免疫细胞化学、western blot表明GDNF基因修饰细胞能正确表达GDNF且基因修饰不影响其增殖与分化。结论建立GDNF基因修饰大鼠胚胎中脑神经干细胞,为进一步应用其开展帕金森病的细胞移植治疗研究奠定基础。Objective To establish glia cell line derived neurotrophic factor（GDNF） gene modified midbrain-derived neural stem cells. Methods The coding sequence of human GDNF was amplified by RT-PCR from U251 cells. By gene recombination technique, human GDNF coding sequence was inserted into eukaryotic expression vector pEGFPN1 which contain EGFP codiiag sequence. The recombinant plasmid pEGFPN1-GDNF was identified with restriction enzyme di- gestion and DNA sequencing. The El4 rat embryonic midbrain-derived neural stem cells were isolated and cultured. The cells of the third passage were transfected with plasmid pEGFPN1-GDNF using FuGENE HD transfection reagent and screened with medium containing G418. The expression of EGFP was observed with fluorescence microscope. The expression of GDNF was analyzed by immunocytochemistry as well as western blot. The differentiation ability of gene modified cells was identified by immocytochemistry research after in vitro differentiation. Results The RT-PCR product of GDNF coding sequence was a 650bp specific segment. By restriction enzyme digestion,the recombinant plasmid vector was diges- ted into 650bp and 4.7kb fragments. The expression of EGFP was initially found 12h after transfection,increased remarkably 24h after transfection and reached a summit at 48h. One month after screened with medium containing G418 ,the positive clones were formed. The immunocytochemistry and western blot showed the GDNF was expressed successfully in cells. In addition, the immunocytochemistry showed the cells were Nestin positive, after differentiation the cells expressed GFAP, β-III-tubulin and TH. Conclusion The GDNF gene modified El4 rat embryonic midbrain-derived neural stem cells were successfully established, which are multipotent and have the ability to self-renew and will provide foundation for the further research about cell therapy of Parkinson＇ s disease.

关 键 词：脑源性神经营养因子基因 重组质粒 中脑神经干细胞 基因转染 

分 类 号：Q343[生物学—遗传学]