GDNF基因修饰的BMSCs向神经元样细胞的分化及神经营养因子的表达

Differentiation of GDNF gene-modified bone marrow mesenchymal stem cells into neuron-like cells and expression of neurotrophic factors

目的:探讨胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)基因修饰的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向神经元样细胞的分化以及神经营养因子的表达。方法:用GDNF重组腺病毒载体和空病毒质粒(blank virus plasmid,BVP)分别感染大鼠BMSCs 2d(称作GDNF/BMSCs和BVP/BMSCs),用免疫荧光染色法检测细胞的神经元特异性标志物微管相关蛋白(microtubule-associated protein 2,MAP2)的阳性细胞数量,PCR法检测细胞MAP2和GAP-43 mRNA的表达,ELISA法检测细胞上清液中GDNF和NGF的表达。结果:BVP/BMSCs组未见MAP2阳性细胞,不表达MAP2和GAP-43 mRNA,GDNF/BMSCs组MAP2阳性细胞率为(42.21±4.79)%,表达MAP2和GAP-43 mR-NA,其上清液中的GDNF和NGF蛋白含量高于空病毒感染组(P<0.05)。结论:GDNF具有促进BMSCs向神经元样细胞分化的作用,其分化作用可能与GAP-43,GDNF和NGF的表达上调有关。

Objective： To investigate the differentiation of GDNF gene-modified bone marrow mesenchymal stem cells into neuron-like cells and the expression of neurotrophie factors. Methods： GDNF recombinant aden- ovirus vector and blank virus plasmid （BVP） were used to infect rat BMSCs, to obtain GDNF-BMSCs and BVP- BMSCs. Immunofluorescence staining was used to observe the number of neuron specific marker, the mircotubule- associated protein 2 （MAP2）-positive cells.RT-PCR technique was used to examine the expression levels of MAP2 and GAP-43 mRNA in cell pellets. ELISA assay was employed to detect the expression levels of GDNF and NGF protein in supernatant. Results： In the BVP/BMSCs group, no MAP2-positive staining was observed and MAP2 and GAP-43 mRNA expression were nearly undetectable. In the GDNF/BMSCs group, MAP2-positive rate was （42.21＋4.79）% and the mRNA of MAP2 and GAP-43 was observed.The expression of GDNF and NGF proteins was higher in the GDNF/BMSCs group than in the BVP/BMSCs group （P〈0.05）. Conclusion： GDNF has the effect of promoting the differentiation of BMSCs to neuron-like cells, and its effect may be correlated with the up-regulation of expression of GAP-43, GDNF and NGF.