Schäfer Karl-Herbert, Hagl Cornelia Irene, Rauch Ulrich
Department of Pediatric Surgery, University Hospital Mannheim, University of Heidelberg, Heidelberg, Germany.
Pediatr Surg Int. 2003 Jul;19(5):340-4. doi: 10.1007/s00383-003-1007-4. Epub 2003 Jul 5.
The enteric nervous system (ENS) derives from neural crest cells, which migrate from the neural tube into the developing gut. The neuronal and glial precursor cells migrate mainly from the oral towards the anal end of the gastrointestinal tract. So far, knowledge about the multipotent influences upon the ENS development, especially its neurotrophic support, derives mainly from knock-out models. The in vitro technique of isolating enteric neuronal precursor cells allows to study the effects of various factors upon their appropriate development in more detail. We therefore adapted the method of growing neurospheres, which are agglomerates of neuronal precursor cells and differentiated neurones and glial cells, from the central nervous system (CNS) for the ENS. The gut of NMRI mice at E12 were dissected, mildly dissociated and plated in 25-cm(2) culture flasks. The cultures were maintained in N1 supplemented DMEM/F12 medium with the appropriate neurotrophin cocktails (bFGF, GDNF, Neurturin, CNTF). After several days in culture most of the cells die, while the surviving cells form clusters from which domes, and later spheres arise. The spheres could be harvested and processed for further experiments. First investigations revealed, that the amount of precursor cells was much less in enteric neurospheres as seen in corresponding cultures from the CNS. We found about 43% HNK-1-NCAM+ in enteric and approximately 90% Nestin-+ cells in midbrain neurospheres. Differentiation studies of the enteric neurospheres showed that especially ciliary neurotrophic factor (CNTF) increased the number of enteric neurones (PGP positive), while the amount of HNK-1 precursor cells decreased under the influence of all tested neurotrophins but GDNF. The culture of the freshly dissociated enteric neurospheres in a three-dimensional matrix yielded a secondary network which allows to investigate the pattern formation of the ENS. The generation of enteric neurospheres and the following differentiation and 3D culture in vitro can increase our knowledge of the amount and time point of neurotrophic as well as the ECM-protein influence upon the appropriate development of the ENS.
肠神经系统（ENS）起源于神经嵴细胞，这些细胞从神经管迁移至发育中的肠道。神经元和神经胶质前体细胞主要从口腔向胃肠道的肛门端迁移。到目前为止，关于对ENS发育的多能影响，特别是其神经营养支持的知识，主要来自基因敲除模型。分离肠神经元前体细胞的体外技术能够更详细地研究各种因素对其正常发育的影响。因此，我们将从中枢神经系统（CNS）培养神经球（神经球是神经元前体细胞以及分化的神经元和神经胶质细胞的聚集体）的方法应用于ENS。解剖E12期NMRI小鼠的肠道，轻度解离后接种于25平方厘米的培养瓶中。培养物在添加了N1的DMEM/F12培养基中，加入适当的神经营养因子混合物（碱性成纤维细胞生长因子、胶质细胞源性神经营养因子、神经营养素、睫状神经营养因子）进行培养。培养几天后，大多数细胞死亡，而存活的细胞形成细胞簇，随后形成圆顶，再后来形成球体。可以收获球体并进行进一步实验。初步研究表明，肠神经球中的前体细胞数量比中枢神经系统相应培养物中的少得多。我们发现肠神经球中约43%的细胞HNK-1-NCAM呈阳性，而中脑神经球中约90%的细胞巢蛋白呈阳性。肠神经球的分化研究表明，特别是睫状神经营养因子（CNTF）增加了肠神经元（PGP阳性）的数量，而在除胶质细胞源性神经营养因子外的所有测试神经营养因子影响下，HNK-1前体细胞的数量减少。将新鲜解离的肠神经球培养在三维基质中可产生二级网络，这有助于研究ENS的模式形成。肠神经球的生成以及随后的体外分化和三维培养可以增加我们对神经营养以及细胞外基质蛋白对ENS正常发育的影响的数量和时间点的了解。
