Objective To investigate the influence of co-culture of microglia and neural stem cells (NSCs) on differentiation from NSCs into dopaminergic neurons in vitro.Methods (1) The microglia from neonatal SD rats was purified and identified.After the NSCs were isolated from 14-day pregnant SD rats,the cells were cultured and identified.(2) The identified NSCs were randomly divided into 2 groups:simple NSCs culture group and NSCs+microglia co-culture group.After the cells in both groups were cultured for 6 days,immunofluorescence assay and Westem blotting were performed to detect the protein expression of TH,DAT and Pitx3,the factors associated with the differentiation and maturity of dopaminergic neurons.(3) PCR was used to detect the gene transcription of TH,DA T and Pitx3 in the cells from the 2 groups and differences were compared between the 2 groups.Results (1)The staining of CD1 1b/c in the microglia mixed cultured in the neonatal SD rats was positive,with a purity of above 95％;the staining of NSCs identified via nestin was positive in the 14-day pregnant SD rats.(2) The immunofluorescence assay showed that the amounts of positive proteins of TH,DAT and Pitx3 in the NSCs+microglia co-culture group were significantly larger than in the simple NSCs culture group (P＜0.05).The Western blotting showed that the protein expression levels ofTH,DAT and Pitx3 in the NSCs+microglia co-culture group were significantly higher than in the simple NSCs culture group (P＜0.05).(3) The PCR detection showed that the gene transcription levels of TH,DA T and Pitx3 in the NSCs+microglia co-culture group were significantly higher than in the simple NSCs culture group (P＜0.05).Conclusion Co-culture of NSCs and microglia via Transwell may promote differentiation from NSCs into dopaminergic neurons.