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目的探讨原代小鼠神经干细胞(NSCs)的培养方法及骨形态发生蛋白-2(BMP-2)对NSCs增殖和分化的影响。方法采用机械吹打结合尼龙膜滤过的方法,从胚胎12.5 d的S129小鼠大脑分离NSCs并培养;用巢蛋白(nestin)免疫荧光染色鉴定NSCs;将NSCs分为对照组和BMP-2处理组(BMP-2终浓度10 ng/mL),培养后进行BrdU染色和微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)免疫荧光染色。结果①镜下观察原代培养的NSCs,胞体圆形,单个或成对存在,3~5 d后形成神经球,神经球90%以上细胞为nestin阳性,台盼蓝染色表明活细胞数大于95%。②BMP-2处理组BrdU阳性细胞比率明显低于对照组(P<0.05)。③BMP-2处理组MAP2阳性细胞比率显著低于对照组(P<0.05),而GFAP阳性细胞比率显著高于对照组(P<0.05)。结论①运用机械吹打结合尼龙膜滤过的方法可以得到存活率高的原代NSCs;②10 ng/mL BMP-2可抑制NSCs增殖及其向神经元方向分化,促进其向神经胶质细胞方向分化。
Objective To investigate the culture method of primary mouse neural stem cells (NSCs) and the effect of bone morphogenetic protein-2 (BMP-2) on the proliferation and differentiation of NSCs. Methods NSCs were isolated from the brains of S129 embryos at 12.5 days and cultured by mechanical pipetting and nylon membrane filtration. NSCs were identified by nestin immunofluorescence staining. NSCs were divided into control group and BMP-2-treated group (BMP-2 at a final concentration of 10 ng / mL). After culture, BrdU staining and MAP2 and GFAP immunofluorescence staining were performed. Results ① The cultured primary NSCs were observed by microscopy. The cell bodies were round, single or in pairs. Neurospheres were formed after 3 ~ 5 days. More than 90% neurospheres were nestin positive. Trypan blue staining showed that the number of viable cells was more than 95 %. ② The ratio of BrdU positive cells in BMP-2 group was significantly lower than that in control group (P <0.05). (3) The ratio of MAP2-positive cells in BMP-2-treated group was significantly lower than that in control group (P <0.05), but the ratio of GFAP positive cells was significantly higher than that in control group (P <0.05). Conclusions ① Primary NSCs with high viability can be obtained by mechanical blowing and nylon membrane filtration. ②10 ng / mL BMP-2 can inhibit the proliferation and differentiation of NSCs into neurons and promote their differentiation into glial cells .