基因工程化分泌神经营养因子-3的鼠胚神经干细胞实验研究

关键词：神经干细胞 基因工程 神经营养因子-3 脊髓损伤

Keywords:Lentivirus  neural stem cell  neurotrophic factor-3  spinal cord injury  genetic engineering

摘要：

      目的：探索以Lentivirus为载体,构建同时表达绿色荧光蛋白（GFP）和神经营养因子-3（NT-3）的基因工程化鼠胚神经干细胞（NSC）的可行性.方法：体外分离培养鼠胚NSC,用同时携带NT-3和GFP的lentivirus转染构建工程化NSC：用荧光显微镜、鼠胚背根神经结培养（Dorsal Root Ganglion,DRG）、Western blot等方法检测基因工程NSC的转基因表达.结果：荧光显微镜观察到几乎100%的工程化NSC表达GFP;DRG培养和Western blot检测到基因工程化NSC能高效分泌NT-3蛋白.结论：以Lentivirus为载体,构建同时携带并稳定表达GFP和NT-3的基因工程化鼠胚NSC是可行的,可为脊髓损伤基础研究提供有价值的细胞资源.

Abstract:

      Objective:To construct the genetic engineering rat neural stem cell(NSC) mediated by Lentivirus to express both green fluorescence protein (GFP) and neurotrophic factor-3(NT-3) at the same time in order to afford the grafts for the further biomedical applications.Method:After the rat NSC was isolated and identified, the rat NSC was genetic modified by Lentivirus to express GFP and rat NT-3; then the transgenic expression was detected by the methods of fluorescence microscope?dorsal root ganglion of fetal rats and Western blot.Result:The genetic engineering NSC was constructed successfully. All of the genetic engineering NSC that expressed bright green fluorescence by the fluorescence microscope was observed. The conditioned medium of transgenic NSC could induce neurite flourishing outgrowth from DRG; The genetic engineering NSC expressed high level NT-3 which could be detected by the methods of Western blot.Conclusion:The genetic engineering rat NSC is successfully constructed,which can be applied widely in the basic study and the further clinic application of the spinal cord injury as neural grafts.