TIMP3激活Shh/Gli通路调控neuroligins基因诱发先天性巨结肠症的作用机制

先天性巨结肠症（HSCR）是小儿先天性消化道常见畸形，因其发病机制不明确，导致诊疗困难。我们前期研究发现纤维粘连蛋白与Neuroligins基因间存有密切地相互调控，证实HSCR的发病机制是“基因异常”和“肠壁微环境”共同作用的结果。预实验发现对肠神经系统突触发生和成熟起重要作用的Neuroligins基因与TIMP3、Sonic hedgehog/Gli（Shh/Gli）通路密切相关。基于此，提出科学假设：“TIMP3可能通过激活Shh/Gli通路，抑制Neuroligins的表达，进而调控肠神经嵴细胞的迁移和定植，从而参与HSCR的发生发展”。本课题利用免疫组化、PCR、干细胞分离培养、病毒转染等分子生物学技术，分别对动物模型、人体标本、细胞进行试验，探讨TIMP3通过Shh/Gli通路调节Nlgns基因诱发HSCR发病机制，为临床基因治疗、胎前干预提供潜在的靶点，探索新的诊疗方法。

Hirschsprung's disease (HSCR) is a common malformation of congenital gastrointestinal tract in children, whose unclear pathogenesis can lead to difficult diagnosis, treatment and poor prognosis. Our previous research found that there is a close mutual regulation between the extracellular matrix fibronectin and Neuroligins (Nlgns) genes, confirming that the pathogenesis of HSCR is the result of the joint action of "gene abnormalities" and " microenvironment changes". Pre-experiments found that the Nlgns gene, which plays an important role in synaptic development, maturation, and function of the enteric nervous system, is closely related to the TIMP3 and Sonic hedgehog / Gli (Shh / Gli) pathways, while the Shh/Gli pathway may be an important signaling pathway connecting extracellular matrix with Nlgns gene. Based on this, a scientific hypothesis is proposed: "TIMP3 can activate the Shh / Gli pathway to cause the down-regulation of the target gene Nlgns, regulate the migration and proliferation of gut neural crest cells, and cause the loss of distal ganglion cells and HSCR." In this study, molecular biological techniques such as immunohistochemistry, immunofluorescence staining, PCR, stem cell isolation, gene transfection and cell apoptosis will be used to conduct experiments on animal models, human specimens and cells at the protein and gene levels, respectively. The Shh / Gli pathway regulates the pathogenesis of HSCR induced by the Nlgn genes, providing potential targets for clinical gene therapy and prenatal intervention, and explores new therapeutic methods.