人胶质细胞源性神经营养因子基因修饰猴雪旺细胞的构建与鉴定  

CONSTRUCTION AND IDENTIFICATION OF RHESUS MONKEY Schwann CELLS MODIFIED WITH HUMAN GLIAL CELL DERIVED NEUROTROPHIC FACTOR GENE

作　　者：巴越洋[1] 王辉[1] 宁昕杰[1] 

机构地区：[1]中山大学附属第三医院神经外科,广州510630

出　　处：《中国修复重建外科杂志》2013年第11期1363-1367,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30901542);广东省自然科学基金资助项目(7301217);广东省医学科学基金资助项目(B2007061);广东省科技计划资助项目(20120314);中山大学高校基本业务费青年教师培育资助项目(12ykpy44)~~

摘　　要：目的构建人胶质细胞源性神经营养因子(human glial cell derived neurotrophic factor,hGDNF)基因修饰的猴雪旺细胞稳定细胞系。方法以pUC19-hGDNF为模板,扩增含hGDNF基因序列,将其插入质粒pBABEpuro,构建pBABE-puro-hGDNF重组真核表达载体,经酶切鉴定及DNA测序鉴定重组质粒。从恒河猴提取、培养、纯化雪旺细胞。包装病毒,利用含hGDNF基因的重组逆转录病毒转染雪旺细胞,实时荧光定量PCR、Western blot检测hGDNF mRNA及蛋白表达情况。结果 PCR产物hGDNF基因编码序列大小约596 bp。重组表达载体经双酶切鉴定,含有1条596 bp的特异性条带,其基因测序结果与基因库中hGDNF基因序列片段相同,提示hGDNF基因成功转入逆转录病毒。逆转录病毒转染的雪旺细胞hGDNF mRNA及蛋白表达量显著高于未转染雪旺细胞,差异有统计学意义(P<0.05)。结论成功构建hGDNF基因修饰的猴雪旺细胞稳定细胞系,能长期稳定高效释放hGDNF,为进一步将其应用于神经损伤修复奠定了基础。Objective To construct the rhesus monkey Schwann cells （SCs） modified with human glial cell derived neurotrophic factor （hGDNF） gene. Methods The coding sequence of hGDNF amplified by PCR from pUC19-hGDNF was inserted into eukaryotic expression vector pBABE-puro. The recombinant eukaryotic expression vector pBABE-puro-hGDNF was identified with restriction enzyme digestion and DNA sequencing. The SCs were isolated from rhesus monkeys, cultured and purified. The SCs were transfected with the recombinant retrovirus vector containing hGDNF gene. The mRNA and protein expressions of hGDNF were analyzed by real-time fluorescent quantitative PCR and Western blot. Results The PCR product of hGDNF coding sequence was a 596 bp specific segment. The recombinant eukaryotic expression vector was digested into a 596 bp specific segment by specific restriction enzyme and another segment. The 596 bp segment confirmed by DNA sequencing was consistent with hGDNF sequence on GenBank. Restriction enzyme digestion and sequencing results showed that the coding sequence of hGDNF was successfully inserted into the recombinant retrovirus vector and the mRNA and protein expressions of hGDNF were significantly higher in transfected SCs than non-transfected SCs （P 〈 0.05）. SCs modified with hGDNF gene are successfully constructed and hGDNF can be released provide a foundation for the further research about cell therapy of hGDNF-SCs in the repair

关 键 词：人胶质源性神经营养因子 重组逆转录病毒 基因转染 雪旺细胞 恒河猴 

分 类 号：R651.3[医药卫生—外科学]