脑源性神经营养因子真核表达载体转染人脐带间充质干细胞  

Transfection of an eukaryotic expression vector expressing brain derived neurotrophic factor into human umbilical cord mesenchymal stem cells

作　　者：孙兆明[1] 袁源[2] 潘树茂[2] 张菊华[2] 王媛丽[3] 汤国太[2] 关茂武[2] 陈鸿光[2] 

机构地区：[1]烟台毓璜顶医院,神经内科,山东省烟台市264000 [2]烟台毓璜顶医院,神经外科,山东省烟台市264000 [3]烟台毓璜顶医院,妇产科,山东省烟台市264000

出　　处：《中国组织工程研究》2012年第45期8413-8418,共6页Chinese Journal of Tissue Engineering Research

基　　金：烟台市科技发展项目资助(2007139-4)~~

摘　　要：背景:干细胞移植是神经损伤修复领域的研究热点,目前多数研究集中于神经干细胞和骨髓干细胞移植研究,而脐带间充质干细胞相关研究目前国内刚刚起步,后者较前者具有来源广泛,伦理限制少等优势。目的:构建脑源性神经营养因子真核表达载体,并将其转染至人脐带间充质干细胞中,建立稳定表达脑源性神经营养因子的细胞模型。方法:应用反转录PCR获得脑源性神经营养因子的基因序列,然后将脑源性神经营养因子的基因序列插入到pcDNAⅢ载体的多克隆位点,构建脑源性神经营养因子蛋白表达载体,并转染脐带间充质干细胞;WesternBlot体外检测携带脑源性神经营养因子基因的脐带间充质干细胞的脑源性神经营养因子蛋白表达水平;取培养好的PC12细胞和胎鼠皮质神经元加入含脑源性神经营养因子的脐带间充质干细胞培养基,检测脑源性神经营养因子蛋白的生物活性。结果与结论:成功构建脑源性神经营养因子真核表达载体,该载体能在脐带间充质干细胞中高水平表达脑源性神经营养因子蛋白,表达的脑源性神经营养因子蛋白体外检测具有生物活性。可见脐带间充质干细胞有可能作为脑源性神经营养因子真核表达载体用于神经损伤修复研究。BACKGROUND： Stem cells transplantation is the research hot spot in the area of nerve injury repair. At present, most researches have focused on neural stem cells and bone marrow stern cells transplantation, and there are rare reports on the research of umbilical cord mesenchymal stem cells, but the umbilical cord mesenchymal stem cells have the advantages of rich source and little ethical restrictions. OBJECTIVE： To construct an eukaryotic expression vector expressing brain derived neurotrophic factor, and to transfect it into the umbilical cord mesenchymal stem cells and establish a cell model of brain derived neurotrophic factor. METHODS： Firstly, the c-DNA of brain derived neurotrophic factor was gained by reverse transcription-PCR and then inserted into multi-clone site of pcDNAⅢ vector to construct the expression vector of brain derived neurotrophic factor pcDNAⅢ. Then the vector was used to transtect umbilical cord mesenchymal stem cells. At last, the expression level of brain derived neurotrophic factor in the umbilical cord mesenchymal stem cells was tested by Western blot; the cultured PC12 cells and the embryo rat pallium neurons were added into the umbilical cord mesenchymal stem cells containing brain derived neurotrophic factor to test the biological activity of brain derived neurotrophic factor. RESULTS AND CONCLUSION： The eukaryotic expression vector expressing brain derived neurotrophic factor was successfully constructed, and this vector could highly express the brain derived neurotrophic factor protein in umbilical cord mesenchymal stem cells. In vitro testing showed the brain derived neurotrophic factor protein had the biological activity. It indicates that the umbilical cord mesenchymal stem cells can be used as the eukaryotic expression vector of brain derived neurotrophic factor for research on the nerve injury repair.

关 键 词：脑源性神经营养因子 脐带间充质干细胞 pcDNAIII载体 PC12细胞 胎鼠皮质神经元 SD大鼠 克隆 

分 类 号：R394.2[医药卫生—医学遗传学]