摘要
目的 构建携带大鼠胶质细胞源性神经营养因子(GDNF)基因的真核细胞表达载体,为应用GDNF进行如帕金森综合征之类的神经元退化性疾病的基因治疗打基础。方法 采用RT- PCR方法从大鼠胎脑组织总RNA中扩增出该基因的c DNA序列,并克隆到增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体p EGFP- C1中,对重组质粒p EGFP- GDNF进一步鉴定。采用电转及阳离子脂质体将重组质粒p EGFP- GDNF转染至SH- SY5 Y细胞。结果 大鼠GDNF c DNA已正确地克隆到真核表达载体p EGFP- C1中,而构建成重组大鼠质粒p EGFP-GDNF。GDNF基因可稳定表达在细胞中。结论 真核细胞表达载体p EGFP- GDNF以及表达GDNF工程细胞SH-SY5 Y的成功构建,为进一步开展GDNF基因治疗PD等中枢神经系统疾病奠定了基础。
Objective To construct a kind of engineered cells that can secrete human glial cell line derived neurotrophic factor(GDNF)and study its possible effects on genetic therapy of Parkinson's disease.Method The cDNA encoding the mature rat glial cell-derived neurotrophic factor (GDNF)was isolated was cloned into eucaryotic expression vector pEGFP-C1 of EGFP reported gene encoding green fluorescence protein in the form of fused protein.By using lipofectin and electric methods,It was transfected into SH-SY5Y cell line.Results The expression vector of recombinant plasmid pEGFP-GDNF was successfully constructed.Using fluorescence microscope analysis demonstrated that the GDNF gene can be transcripted and translated in SH-SY5Y cells.Conclusion It suggests that the engineered SH-SY5Y cells can secrete human GDNF and could play an important role in gene therapy of Parkinson's disease.
出处
《中风与神经疾病杂志》
CAS
CSCD
北大核心
2005年第2期104-106,共3页
Journal of Apoplexy and Nervous Diseases
基金
上海市重点基金资助项目 (0 2 41190 3 7)
上海市引进海外高层次留学人员专项基金资助项目 (2 0 0 4-1-5)
