小鼠胚胎肠神经嵴干细胞的分离培养及鉴定

目的建立分离培养及鉴定小鼠肠神经嵴干细胞(gut neural crest stem cells,GNCSCs)的方法,为临床应用奠定基础。方法分离胚鼠肠管,消化后接种于添加N2、B27和碱性成纤维细胞生长因子的DMEM/F12培养基,贴壁培养;连续传代后用血清促进GNCSCs分化;最后用免疫细胞化学方法染色鉴定。结果分离培养的细胞聚集生长,形成的克隆球可以传代;克隆球p75染色阳性;分化后的细胞分别表达肠神经元、神经胶质细胞和平滑肌细胞特异性抗原。结论分离培养的克隆球具有自我更新和多向分化的能力,表达肠神经嵴干细胞的特异性抗原p75,是肠神经嵴干细胞。

Isolation and culture of neural crest stem cells of embryonic mice in vitro

Objective To establish a satisfactory method of isolating,culturing and determining gut neural crest stem cells,which may provide theoretical basis for clinical application.Methods The guts of embryonic mice removed and dissociated were plated into serum-free DMEM/F12 medium.The mitogen-free DMEM/F12 medium supplementd with 10% fetal bovine serum was used to induce differentiation of GNCSCs.Neurospheres and their derivations were determined with immunocytochemical and immunofluorescent staning.Results Neurospheres were generated in the simplified serum-free medium.The staining results showed that enteric neurospheres were GNCSCs and could differentiate into neurons,glial cells and smooth muscle cells by serum-induction.Conclusion GNCSCs have the capacity of self-renewal and proliferation,and give rise to neurons,glial cells and myofibroblasts.