多聚赖氨酸和明胶对神经干细胞增殖和分化的影响

摘要背景：目前大鼠神经干细胞体外诱导分化的研究报道诸多，但其分化过程很难控制，很多实验的操作方法复杂，分化比率也很低。目的：探索大鼠胚胎前脑神经干细胞体外原代及传代培养方法，并观察其分化规律。方法：胎鼠在无菌条件下分离出前脑，制备单细胞悬液，以1×1011L-1接种于含N2的DMEM/F12培养基中培养，传代培养过程中加入BrdU，标记神经干细胞球。诱导分化实验分为多聚赖氨酸铺板组、明胶铺板组和无铺板组。采用体积分数20%胎牛血清刺激其分化。免疫细胞化学检测nestin、BrdU及在血清诱导条件下神经干细胞向神经细胞分化的能力。结果与结论：细胞呈神经干细胞样生长，具有连续增殖能力，可以传代培养。传代神经球中的细胞均呈nestin阳性和BrdU阳性。多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经细胞能力强于无铺板组(P < 0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P > 0.05)。神经谱系标记物神经胶质纤维酸性蛋白和微管相关蛋白2的免疫细胞化学结果均阳性。结果表明，大鼠胚胎前脑富含神经干细胞，其分化观察，多聚赖氨酸和明胶在诱导神经干细胞分化中作为细胞贴壁支持物提高分化细胞数量的作用，且多分化为星形胶质细胞。关键词：多聚赖氨酸；神经干细胞；明胶；增殖分化；体外培养doi:10.3969/j.issn.1673-8225.2010.47.004

Effect of polylysine and gelatin on proliferation and differentiation of neural stem cells

AbstractBACKGROUND: Currently, there are plenty of the investigations regarding the induced differentiation of neural stem cells (NSCs) in vitro, but the process of differentiation is difficult to control, many methods are complicated to operate, and the proportions of differentiation are very low.OBJECTIVE: To discuss in vitro cultivation of rat embryonic forebrain NSCs and to observe the differentiation rule of NSCs.METHODS: The forebrains were isolated from fetal rats under aseptic conditions, to prepare monocell suspension, and cultured with DMEM/F12 medium containing N2 at the density of 1×1011/L. In the process of cultivating, BrdU was added to mark NSC spheres. The induced differentiation experiment was divided into three groups: polylysine culture plate, gelatin plate and no plate culture groups. 20% volume fraction of embryonic bovine serum was used to stimulate the differentiation. Immunohistochemistry method was applied to detect the level of nestin and BrdU, as well as NSCs differentiation into nerve cells in the serum induction. RESULTS AND CONCLUSION: The cells presented the NSC-like growth and continuous proliferative capacity, they can subculture. Nestin and BrdU were both positive in passaged neurospheres. The inducing differentiation effect of the polylysine culture plate group and the gelatin plate group was greater than no plate culture group (P < 0.01), and the polylysine culture plate group had a little higher percentage of neurons differentiating than the gelatin plate group (P > 0.05). The result of immunohistochemistry was glial fibrillary acidic protein-positive and microtubule-associated protein-2-positive. Rat embryonic forebrain is full of NSCs. The polylysine and the gelatin as cells holder in inducing NSCs differentiation can improve NSCs differentiation and mostly differentiate into the astrocytes through differentiation observation.