神经调节素1调控斑马鱼肠神经系统发育的初步研究

Role of neuregulin 1 in regulating the development of enteric nervous system in zebrafish

目的神经调节素1（neuregulin1，NRG1）可能是先天性巨结肠症的易感基因，本实验选用斑马鱼为模式生物探讨NRG1在肠神经系统发育中的作用。方法克隆斑马鱼NRG1基因，分析多物种NRG1同源性及进化程度；使用原位杂交技术检测NRG1在成年斑马鱼肠道及斑马鱼胚胎中的表达；通过显微注射吗啉代反义寡核苷酸建立斑马鱼NRG1敲低模型，使用原位杂交及免疫荧光技术对比观察斑马鱼表型、肠神经元分化及迁移情况。结果NRG1进化较保守，斑马鱼NRG1与人类NRG1基因有较高同源性；在成年斑马鱼肠道中，NRG1主要位于黏膜层，在肌层和浆膜层均未见表达；胚胎中，NRG1自胚胎早期即开始表达，并表达于胚胎及幼体的肠道，表达谱与肠神经发育时相及位置相符；敲低NRG1后，胚胎出现孵出延迟，头部畸形，身体变短，躯体扭曲等表型；注射NRG1-MO后胚胎存活率为29．1%（196／677），较对照组87．5％（446／510）低；注射NRG1-MO后胚胎异常表型率为21．5%（40／186），较对照组5．5％（23／415）高；敲低NRG1后分化的肠神经元计数（3．33±1．53）个，较对照组（43．00±13．23）个少，而且肠神经元从头端至尾端的迁移过程受阻。结论使用斑马鱼这一模式动物研究NRG1在肠神经发育中的作用，可以间接反映NRG1在人类先天性巨结肠症中的作用，NRG1可能参与斑马鱼肠神经系统发育过程。

Objective To explore the role of Neuregulin 1 （NRG1） in the development of intestinal nervous system. Methods Zebrafish NRGI gene was cloned, NRG1 homology and phylogenetic tree were analyzed in multiple species. In situ hybridization was employed for detecting the expression of NRG1 in adult zebrafish intestine and embryos. Zebrafish NRG1 knockdown model was established by a microinjection of morpholino antisense oligonucleotides. Phenotypes, differentiation and migration of intestinal neuron were shown by in situ hybridization and immunofluorescence. Results Evolutionarily conserved, NRG1 had a high homology between zebrafish and human counterparts. It was expressed in mucosal layer but not muscular layer or serous membrane of adult zebrafish intestine. NRG1 was expressed in larval gut since early embryonic period. The expression spectrum matched enteric nervous development phase. In NRG1 knockdown model, embryonic hatching was delayed. And there were head deformity and short and distorted body. Most importantly, intestinal neuronal differentiation and migration process were inhibited by a down- regulation of NRG1. Survival rate was lower in embryos injected by NRG1-MO than control group [29. 0% （196/677） vs 87. 5% （446/510）]. And the phenotypie abnormality rate was higher than control group[21.5% （40/186）vs 5.5G （23/415）]. Differentiated intestinal neurons in NRG1 knock- down embryos were less than control group [（3. 33 ± 1.53） vs （43.00 ± 13. 23）]. And the anterio- posterior migration of enteric neurons was blocked in NRG1 knock-down embryos. Conclusions An aberrant expression of NRG1 may play an important role in the pathology of HD. The intestinal nervous development in zebrafish is imitated by interfering the NRG1 expression. And a suitable model has been established for studying the mechanism of HD.