基因编辑技术介导下的干细胞治疗Phox2b-/-先天性巨结肠的实验研究

PHOX2B is a key transcription factor that regulates the differentiation of enteric neurons and its mutation is one of the important causes of Hirschsprung disease (HSCR). HSCR is characterized by the totally absence of ganglion in the affected bowel wall. Currently, Resection of the aganglionic segment is the main treatment. But long-term complications seriously affect the patient's physical health and quality of life. So it is urgent to develop new approach to treat HSCR. Enteric neural crest stem cells transplantation may be an effective way. The application of induced pluripotent stem cells (iPSCs) technology and the development of genome editing technology provide us important tools for the use of gene-corrected enteric neural crest stem cells transplantation for the treatment of HSCR. In this study, the iPS cell line with the PHOX2B mutation and enteric neuron differentiation system from pluripotent stem cells have been successfully established in the previous experiment. The purpose of this study was to use CRISPR/Cas9-mediated homologous recombination technology to repair the mutated PHOX2B gene and to compare the enteric neuron differentiation efficiency and their function between the pre- and post-gene editing stages. After the gene correction, the ability of enteric neural crest stem cells engraftment, differentiation, regeneration of the enteric neurons and recovery of the bowel function of HSCR model are detected. This study can not only provide a new idea and technical approach for individualized treatment of HSCR, but also supply scientific basis for exploring the pathogenesis of PHOX2B in HSCR.

PHOX2B是调控肠道神经元分化的关键转录因子，其突变是引起先天性巨结肠（HSCR）的重要原因之一，HSCR表现为病变肠段无神经节，目前以手术治疗为主，但术后长期并发症严重影响患者的身体健康和生活质量，亟需寻找新的治疗手段，而干细胞移植可能是有效的治疗方法。诱导多能干细胞（iPSCs）技术的诞生及基因编辑技术的发展，为我们利用基因修复的干细胞移植治疗HSCR提供了重要工具。本申请团队前期已建立PHOX2B突变的iPS细胞系及肠道神经元分化体系，在此基础上，本研究拟利用CRISPR/Cas9介导的同源重组技术修复突变基因，比较基因修复前后肠道神经元体外分化效率的改变，检测基因修复后移植的干细胞在HSCR模型肠道组织的定植、分化、再生肠神经节的能力和对肠道功能的改善。通过本研究的实施，不仅可以为HSCR的个体化治疗提供新的思路和技术手段，也为探索PHOX2B致HSCR的发病机制提供科学依据。

PHOX2B是调控肠道神经元分化的关键转录因子，其突变是引起先天性巨结肠（HSCR）的重要原因之一，HSCR表现为病变肠段无神经节，目前以手术治疗为主，而干细胞移植可能是有效的治疗方法。本研究利用CRISPR/Cas9介导的同源重组技术修复突变基因，比较基因修复前后肠道神经元体外分化效率的改变，检测基因修复后移植的干细胞在HSCR模型肠道组织的定植、分化、再生肠神经节的能力和对肠道功能的改善。 .本研究目前已建立PHOX2B突变的iPS细胞系，利用CRISPR/Cas9介导的同源重组技术修复突变基因，比较基因修复前后肠道神经元增殖、迁移及体外分化效率的改变。目前实验结果显示实验组及对照组增殖、分化无明显差异，但是神经嵴干细胞的迁移生物特性差异明显。.通过本研究的实施，为探索PHOX2B致HSCR的发病机制提供科学依据。