摘要
目的构建人胶质细胞源性神经营养因子(GDNF)基因的新型真核表达载体,为进行GDNF基因修饰的脐血干细胞移植治疗脑梗死奠定基础。方法将GDNF基因克隆到增强型绿色荧光蛋白(EGFP)真核表达载体中,进行酶切鉴定及DNA测序分析,并将携带有GDNF基因的该真核表达载体,通过脂质体介导,转染人脐血CD34+细胞,荧光显微镜、ELISA检测转基因脐血干细胞GDNF的表达与释放。结果经双酶切鉴定和测序证实已将GDNF基因DNA片段正确插入到真核表达载体中,并能在脐血CD34+干细胞中表达、分泌有活性的GDNF。结论成功构建pEGFP/GDNF基因新型真核表达载体。
Objective To construct the recombinant plasmid of glial cell line-derived neurotrophic factor（GDNF） and confirm in human umbilical cord blood CD34^＋ stem cells and study the value of GDNF as a gene therapy for treatment of cerebral infarction. Methods GDNF was amplified from cortex cells in cerebrum of newborn SD rats by reverse-transcriptase polymerase chain reaction（RT-PCR） and subcloned into the plasmid. The recombinant plasmid was detected by using green fluorescent protein. Then the recombinant plasmid was transfected into human umbilical cord blood CD34 ＋ stem cells and expressed GDNF was detected with RT-PCR. Results Nucleic acid fragment of 640bp was detected by sequencing after GDNF amplification, which was confirmed by homologous comparison. The positive clones were identified by sequencing and endonuclease digestion. The recombinants were screened by RT-PCR. Conclusion The recombinant plamid expressing GDNF is successfully constructed and expressed in human umbilical cord blood CD34^＋ stem cells.
出处
《中风与神经疾病杂志》
CAS
CSCD
北大核心
2008年第6期651-654,共4页
Journal of Apoplexy and Nervous Diseases
基金
国家自然科学基金(No.30572079)
