激活态雪旺细胞源性神经营养因子对胚芽干细胞向神经细胞分化的影响

目的 探索小鼠胚芽干细胞(embryonic germ cells,EGCs)分离、培养的有效方法；观察激活态雪旺细胞(activated Schwann cells,ASCs)源性神经营养因子对小鼠EGCs向神经细胞分化的影响.方法 分离受精11d胚胎小鼠生殖腺嵴及同时取少量的腹壁组织共同消化培养,经胰蛋白酶消化制备成EGCs悬液,将细胞种植在饲养层细胞上.观察小鼠EGCs集落形成情况,并采用阶段特异性胚胎抗原-1染色、碱性磷酸酶染色、过碘酸-雪夫染色对其进行鉴定.采用双酶消化组织块法结合机械分离法分离培养ASCs.神经诱导实验采用基础培养基+小鼠EGCs(空白对照组)和ASCs培养基与小鼠EGCs共培养(实验组),培养3周后对神经诱导结果进行NeuN、MBP、GFAP免疫荧光染色鉴定,计算细胞染色阳性率,对实验结果进行统计学分析.结果 小鼠EGCs呈集落性生长,集落隆起、多数呈鸟巢状、与周围饲养层细胞存在明显界限；集落内的细胞密集,呈现一个或几个核仁、核质比高的圆形或卯圆形；阶段特异性胚胎抗原-1染色、碱性磷酸酶染色、过碘酸-雪夫染色均阳性.小鼠EGCs神经诱导1周后,倒置相差显微镜下可见EGCs克隆的边缘出现向外迁移的细胞,圆形或椭圆形,部分细胞有突起伸出.3周后,迁移的、有细长突起的细胞越来越多,NeuN、MBP、GFAP免疫荧光染色均阳性.细胞染色阳性率比较差异有统计学意义.结论 通过一种简单、经济的方法体外成功分离并获得小鼠EGCs；ASCs源性神经营养因子能够促进小鼠EGCs在体外向神经细胞分化.

Activated Schwann cells-derived neurotrophins induce mouse embryonic germ cells differentiation into neurogenic cells

Objective To seek an optimal method for the separation, culture of mouse embryonic germ cells (EGCs) in vitro, and to observe the influence of Activated Schwann cells (ASCs)-derived neurotrophins on the differentiation capability of mouse EGCs into neurogenic cells. Methodｓ The gonadal ridges and a few abdominal tissues of the 11-day postcoitum (dpc) mouse embryos were isolated and disaggregated by 0.125% trypsin-0.02% EDTA, followed by culture of the mouse EGCs on mouse embryonic fibroblast (MEF) feeders. Monoclonal formation of the mouse EGCs was observed, and the staining of stage specificity embryo antigen-1 (SSEA-1), alkaline phosphatase (AKP), periodic acid-Schiff staining (PAS) were applied to identify the mouse EGCs. Two groups were divided as followed: mouse EGCs+basic medium (control group) and mouse EGCs+ASCs (experimental group). Immunofluorescence(NeuN, MBP, GFAP)analysis was used to evaluate the neurogenic differentiation of mouse EGCs and then to calculate the statistical positive rates of cell staining. All experimental results were analyzed statistically. Resultｓ (1) Identification of mouse EGCs: Mouse EGCs were characterized by a dome-shaped colony containing a large nucleus and a relatively small amount of cytoplasm. All mouse EGCs were positive staining of SSEA-1, AKP, and PAS；(2) The neural induction of mouse EGCs: After one week induction, there were few round or oval cells with long axon-like processes migrating from the edge of the EGCs clones. 3 weeks later, the neurogenic-like cells increased quickly. The results of immunofluorescence(NeuN, MBP, GFAP)staining demonstrated that mouse EGCs could differentiate into neurogenic cells under the influence of ASCs. The positive rate of cell staining was significant. Conclusion In this study, a simple, economical method was applied to successfully separate the mouse EGCs in vitro; mouse EGCs can differentiate into neurogenic cells under the influence of ASCs-derived neurotrophins.