成年小鼠神经干细胞培养、鉴定以及多不饱和脂肪酸含量的测定

摘要

目的:探讨成年小鼠大脑室下区神经干细胞(neural stem cells,NSCs)体外分离培养和鉴定的方法,测定体外培养的神经干细胞多不饱和脂肪酸n6/n3的比例和二十二碳六烯酸(docosahexaenoic acid,DHA)含量.方法:取8～10周龄的C57BL/6小鼠室下区细胞,进行体外悬浮培养,观察NSCs形成情况,免疫荧光鉴定NSCs干细胞特性及体外分化能力,气相色谱鉴定NSCs的多不饱和脂肪酸含量中n6/n3比例及DHA含量.结果:体外培养的细胞可以增殖,并可以连续传代;免疫荧光显示神经干细胞呈抗巢蛋白阳性,神经干细胞在分化培养基培养时可分化为NeuN、TuJ1、GFAP、O4阳性细胞;神经干细胞的多不饱和脂肪酸含量较鼠尾高.结论:采用无血清培养基成功获得成年小鼠室下区的神经干细胞,具有增殖、自我更新和分化的能力;神经干细胞的n3多不饱和脂肪酸含量明显高于鼠尾中的含量,且DHA多不饱和脂肪酸占较高比例.%Objective:To in vestigate the method of culturing neural stem cell s (NSCs) in vitro and measurement of n6/n3 ratios and DHA contents in these NSCs cultured from subventricular zone in adult mice.Methods:Subventricular zone was isolated from the brains of 8～10 weeks old C57BL/6 mice,and the NSCs were cultured in floating in vitro.Some marker genes of NSCs and differentiated cells were detected by immunofluorescendce;polyunsaturated fatty acids and n6/n3 ratios were measured by gas chromatography.Results:NSCs proliferated continuously in vitro,most cells were Nestin positive.NeuN,TuJ1,GFAP and O4 positive cells were detected by immunofluorescence in differentiated neural cells.Polyunsaturated fatty acids were richer in NSCs.Conclusion:NSCs cultured from subventricular zone in adult mice have proliferation and self-renewal abilities.n3 polyunsaturated fatty acids and DHA in NSCs are significantly higher than those in tails.