DCs和NSPCs共培养促进NSPCs增殖及其机制的初步研究

目的观察体外Transwell共培养系统中树突状细胞(dendritic cells,DCs)对神经干/前体细胞(neural stem/progenitor cells,NSPCs)增殖的影响,初步探讨神经营养因子-3(neurotrophin-3,NT-3)在DCs调控NSPCs中的作用机制。方法根据是否采用Transwell小室将DCs和NSPCs共培养分为分隔培养(DCs/NSPCs组)和混合培养(DCs+NSPCs组),ELISA法测定DCs组、NSPCs组、DCs+NSPCs组、DCs/NSPCs组共培养48 h上清中的NT-3的含量。为观察NT-3在DCs调控NSPCs中的作用及可能的机制,成球法检测NSPCs组、NSPCs/DCs+K252a组、NSPCs+DCs组、NSPCs+NT-3组的NSPCs增殖,免疫细胞荧光法和Western blot法检测各组NSPCs表面酪氨酸激酶受体C(TrkC)的表达。结果 ELISA实验结果显示,Transwell共培养48 h上清NT-3的含量,NSPCs/DCs组较DCs组和NSPCs组明显升高(P<0.05)。镜下观察、测量结果显示NSPCs/DCs组和NSPCs+NT-3组神经球数量增多,平均直径明显增大(P<0.05)。免疫细胞荧光和Western blot检测结果表明,NSPCs+DCs组和NSPCs+NT-3组中NSPCs的TrkC表达较NSPCs组和NSPCs/DCs+K252a组高(P<0.05)。结论体外DCs与NSPCs共培养,促进NSPCs增殖、NT-3的分泌及TrkC的表达,NT-3可能是通过TrkC受体参与NSPCs增殖的调控。

Dendritic cells promote neural stem/progenitor cells proliferation in vitro when cocultured

Objective To investigate the effect of dendritic cells(DCs) coculture on the proliferation of neural stem/progenitor cells(NSPCs) in vitro,and explore the role of TrkC receptor and neurotrophin-3(NT-3) in the process.Methods According to whether to adopt the Transwell chamber,the DCs and the NSPCs were co-cultured and divided into separate culture(DCs/NSPCs group) and mixed culture(DCs+NSPCs group,without the use of Transwell chamber).ELISA was used to detect neurotrophin-3(NT-3) protein in the supernatant in 48 h after culture in NSPCs+DCs group(mixed culture),DCs group,NSPCs group,and NSPCs/DCs group(only cells separated by Transwell chamber).Tyrosine kinase receptors C(TrkC) proteins on the surface of NSPCs were detected by immunofluorescence staining and Western blotting analysis in NSPCs group,NSPCs/DCs+K252a group(TrKC blocker),NSPCs+DCs group,and NSPCs+NT-3 group.Results The levels of NT-3 in the supernatant were significantly higher in 48 h after culture in NSPCs/DCs group than in DCs group and NSPCs group(P<0.05).And microscopy showed significantly increases in the number and the average diameter of neurospheres in NSPCs/DCs group(P<0.05).Immunofluorescence staining and Western blotting indicated that the expression level of TrkC were significantly higher in NSPCs/DCs group and NSPCs+DCs group than in NSPCs group and NSPCs/DCs+K252a group(P<0.05).Conclusion DCs in coculture promotes NSPCs proliferation in vitro,which might be through TrkC-NT-3 signal pathway.