小胶质细胞对体外培养神经干细胞向胆碱能神经元分化的影响

Effect of microglia on differentiation from in vitro cultured neural stem cell into cholinergic neurons

背景:神经干细胞的定向分化与小胶质细胞的浓度有关,浓度倍数过小可能达不到应有效应,太大则会产生细胞毒性。目的:观察不同比例的小胶质细胞对体外培养的神经干细胞向胆碱能神经元方向分化的影响。方法:取新生24,48h内Wistar大鼠用于神经干细胞与小胶质细胞的原代培养,免疫组化进行鉴定。神经干细胞与小胶质细胞共同接种于6孔板,接种密度分别为10:1,4:1,1:1,1:4,1:10,每种密度设6孔,以单纯神经干细胞作为对照,共培养3,7,14d,观察细胞的生长情况。结果与结论:原代培养的神经干细胞聚集成球状,悬浮生长,Nestin染色阳性,胞核不着色。小胶质细胞贴壁生长,折光性强,大部分细胞有短的突起呈分支状,小胶质细胞特异性标记抗体CD11b/c染色结果显示细胞纯度在80%以上。与单纯神经干细胞对照组相比,神经干细胞与小胶质细胞以10:1,4:1,1:1,1:4,1:10接种密度共培养组ChAT阳性细胞数均明显升高。共培养组神经干细胞与小胶质细胞比例为4:1时升高幅度显著高于其他接种密度(P<0.05),且在培养第7天时生长达到高峰。结果提示小胶质细胞可以促进神经干细胞向胆碱能神经元方向分化,神经干细胞与小胶质细胞4:1比例共培养第7天时效果最佳。

BACKGROUND：Orient differentiation of neural stem cells depends on concentration of microglia.A small concentration can not play an effect to a certain degree;and a large concentration might cause cytotoxicity.OBJECTIVE：To observe the effect of varying concentrations of microglia on differentiation from in vitro cultured neural stem cells into neurons.METHODS：Newborn Wistar rats within 24 and 48 hours were used for primary culture of neural stem cells and microglia,which were then identified by immunohistochemistry.Neural stem cells and microglia were co-inoculated in 6-well plates,at the density of 10：1,4：1,1：1,1：4,and 1：10,with 6 wells at each density.Neural stem cells alone were considered as control group and incubated for days 3,7 and 14 to observe the cell growth.RESULTS AND CONCLUSION：Primary culture of neural stem cells gathered like a ball and grew in a suspending manner.Nestin staining was positive,but nuclei were not stained.Microglia grew in an adherent manner,with strong refractive index.Most of the cells showed short branch-like protrusions.Microglia-specific marker antibodies CD11b/c staining showed that the cell purity was above 80%.Compared with control group,neural stem cells and microglia cells were co-cultured at a density of 10：1,4：1,1：1,1：4,and 1：1.ChAT-positive cells were significantly increased.If neural stem cells and microglia were co-cultured at the density of 4：1,the increasing was significantly greater than other co-cultured statuses （P0.05）,and the cell growth peaked at 7 days.The microglia could promote differentiation from neural stem cells into cholinergic neurons,and the co-culture of neural stem cells and microglia at a density of 4：1 achieved the best effect at 7 days.