人GDNF在原代培养的大鼠成纤维细胞中的表达  

RECOMBINANT EXPRESSION OF HUMAN GDNF IN PRIMARY FIBROBLASTS OF RATS

作　　者：段德义[1] 杨慧[1] 赵春礼[1] 于凤山[1] 李淑婷 鲁强[1] 徐群渊[1] 

机构地区：[1]首都医科大学北京神经科学研究所,北京100054

出　　处：《解剖学报》2001年第2期101-104,T003,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金! (39780 0 37) ;国家科技攻关计划!(96 -90 1-0 6 -5 1) ;北京市自然科学基金! (79710 0 1) ;北京市教委基金

摘　　要：目的　研究人脑胶质细胞源性神经营养因子 (GDNF)在治疗神经系统疾病中的作用 ,克隆其前体蛋白 c DNA序列 ,并用于真核细胞表达。　方法　提取我国自建的脑胶质瘤 BT32 5细胞总 RNA,用逆转录 PCR法扩增 GDNF全长 c DNA并构建其真核表达载体 p CI- neo- GDNF,然后转染原代培养的成纤维细胞 ,免疫组织化学及原位杂交检测 GDNF在细胞中的表达。　结果　从国人脑胶质瘤细胞中扩增到 5 5 8bp的 GDNF全长 c DNA,并在GDNF转基因原代成纤维细胞中检测到重组 GDNF的转录和表达。　结论　克隆到的 GDNF全长 c DNA片段 ,可用于真核细胞表达 ;其转基因细胞脑内移植后应能治疗包括帕金森病在内的神经系统变性疾病。Objective\ Full\|length cDNA for the precursor protein of human GDNF was cloned and recombinantly expressed in primary fibroblasts in order to investigate the roles of hGDNF in treatment of neurodegenerative diseases. Methods\ Total cellular RNA of glioma BT325,a cell line established from a Chinese patient,was isolated and reverse transcription\|PCR amplification of GDNF full\|length cDNA was performed.The fragment was cloned into plasmid vector pCI\| neo and pCI\| neo \|GDNF was transfected into rat primary fibroblasts.Immunohistochemistry and in situ hybridization were used to detect recombinant GDNF expression in fibroblasts. Results\ A fragment of GDNF,558bp in length,was amplified from reverse transcription mixture of BT325 cell,and recombinant GDNF expression is detected in GDNF gene\|transferred cells. Conclusion\ Full\|length GDNF cDNA,558bp,can be expressed in eukaryotic cells.Implantation of GDNF\|transferred cells into intracerebral tissues can perhaps treat the neurodegenerative diseases such as Parkinson disease.\;

关 键 词：胶质细胞源性神经营养因子 GDNF 逆转录-聚合酶链反应 RT-PCR 基因重组表达 大鼠 

分 类 号：Q784[生物学—分子生物学] Q786