昆明种小鼠胚胎神经干细胞的分离及培养

目的：在神经干细胞的分离培养过程中，国内外的报道多数是用胰蛋白酶对组织细胞进行消化，但是消化时间比较难把握。采用胰酶消化与机械分离法相结合的方式对昆明种小鼠胚胎脑神经干细胞进行分离、培养和初步的免疫组织化学检测，为相关研究建立实验条件。 方法：实验于2006—10／2007-09在广西医科大学基础医学实验室完成。实验室为广西重点实验室、基础医学博士后工作站。①实验材料：孕14-16d的昆明种小鼠由广西医科大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：分离昆明种小鼠胎鼠的脑组织，经胰酶消化加机械吹打后，在加入碱性成纤维细胞生长因子和表皮细胞生长因子的B27无血清培养基中培养。③实验评估：用免疫细胞化学方法鉴定分离的神经干细胞。 结果：在无血清DMEM／F12培养液中，加入碱性成纤维细胞生长因子、表皮细胞生长因子及B27的条件下，培养24h后可见细胞以悬浮方式生长，三五聚集成团块，48h后形成由十数个至数十个细胞组成的，大小不等的细胞球，形态规则，细胞无突起，形成典型的神经球，可传代。免疫细胞化学法检测显示，细胞表达神经巢蛋白。 结论：①来自昆明种小鼠胎脑的神经干细胞能在体外培养、增殖并具有增殖传代能力。②无血清B27培养基添加表皮细胞生长因子、碱性成纤维细胞生长因子有利于神经干细胞的体外培养和促进其增殖。

Isolation and culture of neural stem cells from Kunming mouse embryo

AIM： During the procession of isolating and culturing neural stem cells, trypin was used to digest tissues and cells on the most report at home and abroad, but it was difficult to control the digestive time. We combined trypsinization with mechanical isolation to isolate, cultivate and identify neural stem cells from fetus brain of Kunming mice by the immunohistochemical method, and than set up experimental condition for correlative empirical study.
METHODS： Experiments were carried out in the basic medical laboratory of Guangxi Medical University from October 2006 to September 2007. The lab got the key laboratories of Guangxi and postdoctoral workstation. ①Pregnant 14 to 16 days Kunming mice were provided by the Laboratory Animal Center of Guangxi Medical University. The animal＇s disposal was complied with the principles of animal ethics. ②Brain tissues were isolated from Kunming mice foetus, and blown mechanically after trypsinization. Then the residue was cultured in serum-free medium containing B27, basic fibroblast growth factor and epidermal growth factor. ③ Immunohistochemical method was performed to identify the cultured neural stem cells.
RESULTS： After incubation in serum-free DMEM/F12 medium added with B27, basic fibroblast growth factor and epidermal growth factor for 24 hours. The cells were suspensions with the manner of little neurospheres. After incubation of primary cells for 48 hours, the cultured cells grew much larger and formed typical neurospheres, no marked apophysis and survived well after passaged. Meanwhile, immunocytochemical method showed that these cells showed nestin-positive.
CONCLUSION： ①Neural stem cells from fetus brain of Kunming mice can survive, proliferate and passage well. ②Serum-free B27 medium with basic fibroblast growth factor and epidermal growth factor can benefit to the culture and promote the proliferation of neural stem cells in vitro.