GDNF在人脑胶质瘤细胞中表达的检测及其cDNA的克隆  

RT-PCR ANALYSIS OF GDNF EXPRESSION IN GLIOMA CELL AND cDNA CLONING OF PRECURSOR PROTEIN AND MATURE PEPTIDE OF GDNF

作　　者：段德义[1] 孙成三[1] 杨有文[1] 于凤山[1] 张进禄[1] 徐群渊[1] 

机构地区：[1]首都医科大学北京市神经科学研究所,北京100054

出　　处：《神经解剖学杂志》2001年第1期6-10,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金! (No.39780 0 37) ;国家重点基础研究发展规划项目!<脑功能和脑重大疾病的基础研究>(G19990 5 40 0 8) ;

摘　　要：为研究人脑胶质细胞源性神经营养因子在脑胶质瘤中的表达及其在治疗神经系统疾病中的作用 ,本研究克隆了其前体蛋白及其成熟多肽的 c DNA序列 ,将之用于真核及原核细胞表达。提取我国自建的脑胶质瘤细胞系 BT32 5总 RNA,设计两对引物用逆转录 PCR法扩增上述两种长度的 c DNA片段 ,然后重组于 p GEM-T载体 ,酶切鉴定插入片段正确后测序。结果表明 ,脑胶质瘤细胞中只扩增出 5 5 8bp的胶质细胞源性神经营养因子全长 c DNA片段 ,未见 6 36 bp片段 ;且扩增效率较成熟多肽 c DNA甚低。提示 ,该细胞合成的胶质细胞源性神经营养因子可能还存在不含有信号肽而只能在细胞内潴留的成熟多肽形式。作为自泌性生长因子 ,它可能调节瘤细胞生长等生物学行为 ;同时克隆到的胶质细胞源性神经营养因子全长及其成熟多肽的 c DNA片段 。In order to investigate the expression of GDNF in human glioma cell line and the roles of hGDNF in treatment of nervous system diseases, the cDNA for the precursor protein and the mature peptide of human GDNF were cloned. Total cellular RNA of glioma BT325, a cell line established from Chinese patient, was isolated and reverse transcription PCR amplification of two kinds of cDNA fragments mentioned above was performed. The fragments were then cloned into pGEM-T vector and the inserts sequenced after analysis of the recombinant pGEM-T plasmids by restriction enzymes. The results indicated that the two fragments, 558 bp and 405 bp, were amplified from reverse transcription mixture, while the other expected full-length one, 636 bp, was not obtained. Moreover, amplification efficiency of 558 bp is much lower than that of 405 bp. It was suggested that some of the synthesized GDNF in glioma cell line is perhaps retained within cells because of lack of signal peptide and it, as an autocrine growth factor, might modulate cell biological behaviors such as growth, etc. Full-length cDNA, 558 bp, and cDNA for mature peptide of GDNF were cloned and can be expressed in eukaryotic and prokaryotic cells, respectively.

关 键 词：胶质细胞源性神经营养因子 基因克隆 RT-PCR DNA序列分析 胶质瘤细胞 

分 类 号：R338[医药卫生—人体生理学]