稳定表达NGF、BDNF和NT3星形胶质细胞系的建立及其鉴定

Construction and identification of astrocyte lines expressing NGF, BDNF, and NT3 steadily

目的 :探讨星形胶质细胞直接作为转基因靶细胞进行目的基因的表达。方法 :克隆大鼠 NGF、BDNF和 NT3基因 ,构建真核表达载体 ;3种重组载体分别转染星形胶质细胞 ,G4 18筛选后获得阳性克隆细胞进行扩大培养 ;取基因修饰星形胶质细胞培养液上清培养 PC12细胞或 Trk B- PC12细胞 ;用 Western blotting杂交或免疫组织化学方法检测基因靶细胞目的基因的表达及其水平。结果 :经 G4 18筛选后 ,转染 NGF、BDNF和 NT3基因修饰星形胶质细胞呈 G4 18抗性 ;转染 NGF和 NT3基因条件培养液上清培养的 PC12细胞 ,数量增多 ,突起明显变长 ,转染 BDNF基因条件培养液上清培养的 Trk B- PC12细胞 ,同样数量增多 ,突起明显变长。大部分 G4 18抗性的基因靶细胞出现 NGF、BDNF和 NT3免疫染色阳性。Western blotting杂交显示有相应分子量大小的蛋白条带出现。结论 :星形胶质细胞直接作为基因靶细胞能有效地表达和分泌有生物学活性的 NGF、 BDNF和 NT3。

Objective To study the gene expression of astrocytes as the gene target cells. Methods The NGF, BDNF, and NT3 genes of rats were cloned, the eukaryote expression vectors were established, the three kind of recombinant vectors were used to transfect astrocytes, the positive cloned cells were cultured dilatedly after G418 sifting; using supernates of culture liquid of astrocytes modified by gene to culture PC12 or TrkB-PC12, the expression and its level of gene target cells aimed genes were measured by Western blotting or immunohistochemical method. Results After G418 sifting, astrocytes modified by gene transfected by NGF, BDNF, and NT3 gene showed G418 positive; PC12 cultured in supernate culture liquid transfected by NGF and NT3 showed increases in the number and longer proptosis, TrkB-PC12 cultured in supernate culture liquid transfected by BDNF also showed increases in the number and longer proptosis; largely G418 positive gene target cells showed immune staining positive. Western blotting showed a protein belt with corresponding molecular weight. Conclusion Astrocytes can effectually express and secrete NGF, BDNF, and NT3 with biological activties as gene target cells.