肠神经胶质细胞GDNF-GFRa1/RET自分泌环路在溃疡性结肠炎中的作用及机制

Enteric glial cell (EGC) play a critical role in maintaining the intestinal mucosa barrier integrity and supporting the enteric neurons. Our previous study indicated that the number of EGC and expression of GDNF secreted by EGC was highly increased in inflammatory colon in mice with DSS induced colitis, meanwhile, the increased GDNF protected the colonic epithelium from apoptosis via activation of GFRa1/RET compound receptors and then maintained the barrier integrity, also GFRa1/RET compound receptors were expressed in EGC in UC. Recently, a study showed that EGCS protected themselves from apoptosis via GDNF-GFRa1/RET autocrine loop in Crohn's disease. We hypothesize that GDNF-GFRa1/RET autocrine loop may exist in EGC in inflammation procession of UC and involve in protection of EGC themselves from apoptosis as well as supporting intestinal epithelial cells and neurons. At present study firstly we will investigate whether there exists apoptosis of EGC in colonic tissues of UC mice, then observe the dynamic change of the number , secretion function and apoptosis in EGC in inflammation procession in UC；furthermore, isolated and cultured mice EGC will be stimulated with selected pro-inflammatory cytokines to identify the exist of GDNF-GFRa1/RET autocrine loop to prevention of apoptosis of EGC pre-treated with antibody to GDNF or RET, and the mechanism of its anti-apoptosis is investigated. Finally, the culture supernatant of EGC pre-treated with antibody to GDNF or RET is used to culture intestinal epithelial cell, enteric neuron and treat the mice with DSS-induced colitis by intraperitoneal injection in order to investigate the other functions and action mechanisms of EGC. The aim is to elucidate the action mechanism and clarify the role of EGC in onset and development of UC, provide experiment basis for finding novel therapeutic strategies to UC. Key words: enteric glial cell; dextran sulfate sodium; ulcerative colits; glial cell-derived neurotrophic factor

我们前期研究表明肠神经胶质细胞（EGC）及其分泌的胶质细胞源性神经生长因子（GDNF）在溃疡性结肠炎（UC）时显著升高， GDNF通过活化GFRa1/RET复合受体抗肠上皮细胞凋亡，且UC时EGC上也表达GFRa1/RET复合受体。最近研究显示，CD时EGC通过GDNF-GFRa1/RET自分泌环路抗其凋亡。我们推测在UC发病中，EGC中也存在GDNF-GFRa1/RET自分泌环路并抗其凋亡，且该环路可能参与EGC对上皮细胞，神经元调控。课题将首先观察UC肠组织是否存在EGC凋亡及凋亡数的动态变化；再分离培养小鼠EGC，在细胞水平证实是否存在该自分泌环路并具抗凋亡作用；最后将经GDNF或RET抗体处理的EGC培养上清液腹腔注射结肠炎小鼠或培养肠上皮细胞、肠神经元，探讨该环路的其他作用与机制。旨在阐明EGC在UC发病中的作用与机制，为深入理解UC的发病机制，开辟新的治疗策略提供理论依据。

本项目前期研究表明肠神经胶质细胞（EGC）及其分泌的胶质细胞源性神经生长因子（GDNF）在溃疡性结肠炎（UC）时显著升高，GDNF通过活化 GFRa1/RET复合受体抗肠上皮细胞凋亡，且UC时EGC上也表达GFRa1/RET复合受体。有研究显示CD时EGC通过GDNF-GFRa1/RET自分泌环路抗其凋亡。因此，我们推测在UC发病中，EGC中也存在GDNF-GFRa1/RET自分泌环路并抗其凋亡，且该环路可能参与 EGC 对上皮细胞，神经元调控。本课题在前期研究基础上通过体外培养大鼠EGC细胞株，验证其是否存在该自分泌环路并具抗凋亡作用；本课题组反复应用不同方法均未检测到EGC分泌GDNF，无论是否应用炎性细胞因子刺激，而在LPS刺激下EGC胞内可检测到GDNFmRNA。项目资助发表中英文论文8篇，其中SCI论文2篇。培养硕/博士研究生6名，其中5名已经取得硕士学位，博士研究生1名在读。项目投入经费73万元，支出35.7万元，各项支出均未超出预算。结余经费37.3万元，剩余经费计划用于本项目后续研究支出。