摘要
目的:建立一种简便的分离与鉴定胚胎小鼠肠神经胶质细胞的方法。方法:分离14~16 d胚胎小鼠肠道组织,制备单细胞悬液,用神经胶质细胞生长专用的培养基进行培养,在倒置显微镜下观察胶质细胞的生长情况。在细胞贴壁达90%以上时进行纯化并传代,待细胞贴壁后使用GFAP、S100b和Sox10蛋白抗体作为胶质细胞的特异性标记物用免疫荧光法进行EGCs纯度的鉴定。结果:培养24 h部分细胞贴壁,随培养天数延长贴壁细胞的数量逐渐增多,胞体较前饱满,突起进一步向外延展。12~14 d时细胞贴壁达90%以上,传代后细胞状态良好。免疫荧光染色可见其GFAP、S100b和Sox10蛋白质染色阳性率均达90%以上,鉴定为肠神经胶质细胞。结论:我们建立了一种简便的分离与鉴定胚胎小鼠肠神经胶质细胞的方法,今后可以用于EGCs自身功能及与其他肠道细胞相互作用的研究。
Objective: To develop a simple method to isolate and identify enteric glial cells(EGCs) of embryonic mouse in vitro. Methods: Guts of E14-16 were dissociated and made single cell suspension,which were placed into an optimal growth media for long term culture,observing the changes under a phase-contrast microscope. Glial cells were purified and passaged until the cell attachment rate was up to 90%. Purity of cultures was assessed by immunocytochemistry using antibodies against glial fibrillary acidic protein(GFAP),S100 b and Sox10. Results: Some cells adhered to the wall after a 24 h culture period. More cells firmly attached to the plate with larger cell bodies and longer processes. EGCs were in good conditions after passaging cells on the 12 th-14 th day. More than 90% of DAPI positive cells were GFAP,S100b,and Sox10-immunoreactive,identified as enteric glial cells. Conclusion: We have developed a simple method to isolate and identify EGCs from the gut of embryonic mice,which could be used to define the role of EGCs and to their complex interaction with other cells of the intestine.
作者
李娜
高慧
张煜鑫
李岩松
袁伟
李爽
王强
Li Na;Gao Hui;Zhang Yuxin;Li Yansong;Yuan Wei;Li Shuang;Wang Qiang(Department of Anesthesiology,The First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China)
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2019年第6期636-640,共5页
Chinese Journal of Neuroanatomy
基金
陕西省自然科学基金(2017JM8048)
