先天性巨结肠患儿肠神经干细胞培养的实验研究  

Experimental study of culturing enteric neural stem cells from gut in children with Hirschsprungs disease

作　　者：胡书奇 岳雷[1] 夏肇波 朱利斌[1] 李仲荣[1] 

机构地区：[1]温州医科大学附属第二医院育英儿童医院小儿外科

出　　处：《中华小儿外科杂志》2015年第11期846-850,共5页Chinese Journal of Pediatric Surgery

基　　金：浙江省自然科学基金（编号：Y207272）,浙江省医药卫生平台研究计划重点项目（编号：2012ZDA038）,温州市高层次人才创新技术重点项目资助

摘　　要：目的探索从人类先天性巨结肠患儿的扩张段肠管提取肠神经干细胞的可行性，为进一步开展先天性巨结肠的神经干细胞自体移植治疗研究提供依据。方法利用1例先天性巨结肠手术切除的扩张段肠管，进行酶消化后，制成单细胞悬液，经体外培养形成神经球，并通过CCK8法检测肠神经干细胞在24h、48h、72h、96h、120h和144h六个时间点的OD值，绘制生长曲线，记录增殖情况，通过Nestin、GFAP和TUJ-1细胞免疫荧光化学染色对肠神经干细胞及其分化而来的神经元和神经胶质细胞进行鉴定。结果来自扩张段肠管的单细胞悬液通过原代体外培养10d，可见神经球形成；肠神经球随时间逐渐变大，CCK8法观察肠神经干细胞的0D值随时间而逐渐增高；形成的神经球可以继续传代，经细胞免疫荧光化学染色为Nestin染色阳性，并可分化为神经胶质细胞（GFAP染色阳性）和神经元细胞（TUJ-1染色阳性）。结论能从人类先天性巨结肠患儿的扩张段肠管提取具有自我更新能力的肠神经干细胞，可用于先天性巨结肠的神经干细胞自体移植治疗研究。Objective To explore the possibility of isolating and culturing in vitro enteric neural stem cells from gut in children with Hirschsprung＇s disease （HD） and provide experimental rationales for treating HD with neural stem cell transplantation. Methods Human gut samples were obtained from one child aged 11 months after pull-through surgery due to HD. After enzyme digestion, single ceil suspensions were isolated from postnatal human myenteric plexus. The cells were seeded into serum-free medium and generated neurosphere-iike bodies in vitro. Cell proliferation was observed by the curve of optic density （OD） from CCK8 test at the timepoints of 24, 48, 72, 96, 120 and 144h and growth curve diagram plotted. And neurospheres and differentiated ceils with immunofluorescent staining of nestin were identified by neuron-specific beta III tubulin （TUJ-1） and glial fibrillary acidic protein （GFAP）. Results Single cells from colonic muscle formed neurospheres after culturing for 10 days and these neurospheres could be subcultured and maintained for several weeks in vitro. The OD values of CCK8 test increased progressively. Existing neurospheres, identified as nestin positive cells, could be passaged and differentiated into glial cells （GFAP positive） and neurons （TUJ-1 positive）. Conclusions Enteric neural stem cells may be obtained from human postnatal gut in patients with HD. And it is a potential source of autologous neural stem cells for stem cell transplantation therapy.

关 键 词：巨结肠 先天性 肠神经干细胞 干细胞移植 

分 类 号：R725.7[医药卫生—儿科]