新激素受体GPR54在胸腺细胞发育分化中的功能及机理研究

The development of central lymphoid organ thymus, and differentiation and maturation of T lymphocytes are critical for the immune system. GPR54, a new hormone receptor, has been revealed to play a pivotal role for the onset of puberty. Since thymus development is closely related to puberty, we speculate that GPR54 might participate in thymocyte development and maturation, which has not been reported previously. Our primary data show that GPR54 is extensively expressed in normal thymus. When compared with wild type mice, GPR54 knockout mice have significantly larger thymi, and lower cell density in medulla. Additionally, there are more CD4/CD8 double-positive T cells and less single-positive mature T cells in GPR54 knockout mice. Based on above observations, we are going to investigate the function of GPR54 in thymocyte development mainly use GPR54 knockout mice, together with immune disease models, in vitro cell differentiation, histological and FACS analysis. Experiments are designed to study whether there are any changes in immune responses, whether the lower cell density in medulla is caused by less single-positive T cells, and in which stage does GPR54 affect thymocyte development. We will also use autoimmune disease models to examine whether there are any defects in thymocyte negative selection and its corresponding signaling pathway. Results of this project can further reveal the molecular mechanism of T-cell development and maturation in thymus.

中枢免疫器官胸腺的正常发育以及T细胞在胸腺内的分化成熟对机体免疫系统至关重要。新激素受体GPR54 已被证明能调控青春期的正常启动，而胸腺发育又与青春期发育密切相关，因此GPR54很可能参与调节胸腺细胞分化，迄今为止还没有这方面的报道。申请人初步研究表明，GPR54在正常胸腺中有丰富表达，而且GPR54 基因敲除小鼠的胸腺显著增大，髓质内细胞密度下降，胸腺细胞中CD4+CD8+ 细胞比例增高，CD4或CD8 单阳性成熟细胞显著减少。本课题将主要利用GPR54基因敲除小鼠，从体内体外研究GPR54在胸腺细胞发育分化中的功能，分析髓质内细胞密度下降是否由于成熟性胸腺细胞减少所至，用自身免疫疾病模型探讨GPR54敲除小鼠的胸腺细胞分化是否存在CD4+CD8+阶段的阴性选择失调。进一步研究GPR54调节胸腺细胞分化的分子机理和信号转导途径。本课题的结果将为T细胞在胸腺内分化与成熟机制提供理论补充。

GPR54 在中枢神经系统和胎盘组织中高表达。已有的研究表明它在青春期发育中起着关键的作用，但作为一个新型的激素受体，GPR54在免疫系统中也有广泛的表达，提示可能参与免疫调节。我们的课题研究了GPR54在胸腺发育、T细胞分化和功能、免疫耐受等多方面的生物学功能，发现GPR54的缺失在成年小鼠中导致显著的胸腺体积增大、胸腺细胞增多以及胸腺微结构的变化，而流式细胞分析发现在青春期前的小鼠中，GPR54的敲除引起Treg细胞比例下降。另外伴随着胸腺中凋亡的上升，胸腺细胞亚群中出现CD4单阳性细胞下降和CD4 CD8双阳性细胞上升。这些细胞方面的表型在GPR54缺失的骨髓细胞移植小鼠中得到了肯定。研究发现青春期前KO小鼠胸腺中CCL19在mRNA和蛋白水平都显著下降，说明GPR54对胸腺细胞发育的调节与胸腺中趋化因子表达有关。此外，我们还发现缺失GPR54导致小鼠外周以及胸腺中的T细胞在ConA刺激下比WT小鼠的T细胞产生更强的活化和增殖反应。与之相应的，在完整小鼠、去势小鼠以及骨髓移植小鼠三个体系中，GPR54敲除都诱发更严重的实验性自身免疫性脑脊髓炎（EAE），说明GPR54的缺失导致小鼠自身免疫性的显著上升，而且这种作用可以不依赖于青春期发育和性激素，而通过传统激素途径以外的其他途径介导。这些研究结果揭示了GPR54除通过经典性激素调节免疫系统外，也有不依赖性激素的调节T细胞和免疫耐受的崭新功能。