人GDNF基因的腺病毒载体构建及其在人神经干细胞中的表达

Estabilshment and expression of recombinant human glial cell line-derived neurotrophic factor in human neural stem cells

目的:观察构建的含人胶质细胞源性生长因子(GDNF)基因腺病毒载体对人神经干细胞的感染及其基因表达情况,为脊髓损伤的基因及神经干细胞治疗提供前期实验依据。方法:从12周龄流产人新鲜胚胎中提取人神经干细胞并进行培养,行Nestin免疫荧光染色进行检验。将全长558bp编码人GDNF的cDNA克隆到重组腺病毒载体质粒,并在人胚肾细胞(HEK293细胞)中包装出含有目的基因hGDNF的腺病毒,然后用该腺病毒感染人神经干细胞,应用荧光显微镜和Western-blot检测病毒感染及外源基因的表达情况,并进行GFAP和Tubulin免疫荧光染色检测神经干细胞向神经细胞分化情况。结果:Nestin免疫荧光染色显示所培养细胞为阳性染色红色,表明其具有神经干细胞性状;感染48h后观察到神经干细胞中有大量的增强绿色荧光蛋白(EGFP)表达,以及hGDNF蛋白高表达;感染后的神经干细胞有长的伪足伸出,呈GFAP和Tubulin染色阳性,表明促进了神经干细胞向神经元的分化。结论:腺病毒对神经干细胞具有较高感染效率,可作为一种良好的基因导入载体,实现外源基因hGDNF在神经干细胞内的有效表达,并可为神经干细胞分化为神经元提供更有利条件。

Objective：To investigate the interference and expression of human glial cell line-derived neurotrophic factor（hGDNF） gene in neural stem cells and to evaluate the roles of hGDNF in the genetic treatment of spinal cord injury.Method：Full-length GDNF cDNA,558bp,was inserted into the,early 1 region of adenovirus genomic DNA and immediated by the human cytomegalovirus （gene promoter/enhancer）,and these adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo,then they were used to infect human neural stem cells.The infection and expression of gene were tested under flow cytometry,immunofluorescence and Western-blot after 48 hours.Result：Almost all the cultured cells showed the nestin immunofluorescence positive staining,which was the characteristics of neural stem cell.A great quantity of EGFP was observed and high level of GDNF protein was found,expressed in human neural stem cells infected by recombinant GDNF-adenovirus.After transfection of GDNF gene,many neural stem cells show GFAP and Tubulin immunofluorescence positive staining,which meant that most NSCs differentiated into neuron at that condition.Conclusion：The infective efficiency of adenovirus is greatly acceptable to neural stem cell,thus adenovirus provide a useful vector for exogenous GDNF gene expressed in neural stem cells,which is useful for differentiation of neural stem cell.