摘要
目的联合应用简化无血清培养基和连续传代法，从胚胎14～17d、新生1d、新生5d小鼠肠管分离培养肠神经嵴干细胞（gut neural crest stem cells，GNCSCs），观察其体外培养过程中增殖、分化的特点，用p75、GFAP、Peripherin、α-Actin作为特异性标志来鉴定GNCSCs及其分化的细胞系，比较胚胎和新生小鼠GNCSCs在生长速度、分化细胞的种类及数量上的差异。方法取3组小鼠的肠管，分离制成单细胞悬液，接种于DMEM／F12完全培养基贴壁培养，在连续传代培养中观察克隆球的形成；将克隆球接种于含血清培养基，观察其分化现象；用免疫细胞化学和免疫荧光法检测克隆球及其分化细胞系特异性标志物的表达。结果部分胚胎和新生小鼠肠管细胞在无血清培养基中连续传代培养形成克隆球。在血清刺激下克隆球可分化为多种形态的细胞。克隆球和分化细胞系的免疫染色均为阳性。结论胚胎和新生鼠肠管内存在具有自我更新能力的GNCSCs，且均可分化为神经元、神经胶质细胞和平滑肌三类细胞。新生鼠较胎鼠GNCSCs生长速度慢，分化的神经元数量减少，神经胶质细胞数量增加，平滑肌细胞无显著变化。
Objective To establish a simplified culture system to isolate and passage gut neural crest stem cells （GNCSCs） from embryonic and postnatal mouse, and to compare the difference of growth rate and differentiation of GNCSCs between embryonic and postnatal mice. Methods The gut of mouse in three groups were dissociated and triturated into single cells which were seeded into serum-free DMEM/F12 medium, and the formation of neurospheres was observed during the continuous passage. After adding 10% fetal bovine serum, the differentiation of neurospheres was observed. Immunocytochemistry and immunofluorescence staining were then performed to observe the expressions of p75,GFAP,Peripherin and α-Actin. Results A minority of gut cells formed neurosphere-like cells, and neurospheres generated cells of different types, p75-positive neurospheres and positive derivatives were detected in the experimental group. Conclusions The capacity of self-renew and proliferation of GNCSCs remains in the embryonic and postnatal mouse gut. GNCSCs can differentiate into neuron, glia cell and smooth muscle cell. There are slower growth rate, less neuron, and more neural gila cells in postnatal mice than those in the embryonic mice.
出处
《中华小儿外科杂志》
CSCD
北大核心
2007年第2期73-77,共5页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金（30571932）
关键词
肠神经嵴干细胞
细胞培养技术
细胞分化
巨结肠
先天性
小鼠
Gut neural crest stem cells
Cell culture techniques, Cell differentiation
Hirschsprung disease
Mice
