胰淀素强化调节性T细胞功能的机制

Amylin is co-produced and co-secreted with insulin in the βcells of the pancreatic islets and consequently modulates glucose metabolism. Patients with diabetes mellitus are characterized by amylin deficiency. Recently, our original findings of human amylin as a key immune-regulatory pepetide are as follows: ① amylin induced the expansion of CD4+CD25+ regulatory T cells (Treg) from human peripheral blood mononuclear cells; ② amylin inhibited the proliferation of effector T cells; ③ treatment with amylin decreased the incidence of autoimmune diabetes in non-obese diabetic (NOD) mice; and ④ mice transgenic for human amylin had enhanced Treg function. We hereby propose to further explore the amylin-enhanced Treg function and underlying mechanisms. Patients with newly diagnosed type 1 or type 2 diabetes and healthy subjects will donate their peripheral blood samples to validate the correlation between amylin concentration and Treg count. Then we will perform mainly ex vivo T cell culture, followed by flow cytometry (FACS), enzyme linked immunosorbent assay (ELISA) and Western blot (WB), to prove that human amylin activates Treg to suppress the secretion of cytokines interferon-γ(IFN-γ) and tumor necrosis factor-α (TNF-α) by effector T cells (CD4+CD25- T cells and CD8+ T cells). Mechanisms underlying the amylin-enhanced Treg function involve Toll-like receptors (TLR-2,TLR-4), amylin receptors (RAMP1, RAMP2, RAMP3), cytokines (TGF-β,IL-10) and intracellular PI3K/Akt/NF-κB signaling pathway. The amylin-induced enhancement of Treg function is crucial for protecting islet β cell function by attenuating the autoimmune destruction and proinflammatory state in diabetes mellitus and islet transplantation.

胰淀素与胰岛素由胰岛β细胞共同合成与分泌，调节糖代谢；糖尿病人胰淀素缺陷。我们新发现人胰淀素具有重要免疫调节功能：①诱导人外周血单个核细胞转化为调节性T细胞（Treg）；②抑制效应T细胞增殖；③降低小鼠自身免疫性疾糖尿病的发病率；④强化转基因小鼠Treg的功能。因此，我们将利用新诊断的1型、2型糖尿病患者和健康人外周血T细胞，进一步明确糖尿病人胰淀素与Treg的缺陷及两者相关性；应用活体外T细胞培养、流式细胞仪、酶联免疫吸附和蛋白印迹，深入研究人胰淀素通过Treg抑制效应T细胞分泌IFN-γ和TNF-α的功能。调节机制包括Toll样受体（TLR-2、TLR-4）、胰淀素受体、细胞因子（TGF-β、IL-10）以及Treg内PI3K/Akt/NF-κB信号传导通路。明确胰淀素强化Treg的免疫调节功能及其内在机制，旨在保护胰岛细胞，改善糖尿病和胰岛移植过程中的自身免疫损伤和促炎状态。

本项目主要探讨人胰淀素（human amylin: hA）的免疫调节功能。临床应用领域涉及1型糖尿病（T1DM）、2型糖尿病（T2DM）、胰岛移植以及其它由于调节性T细胞缺乏或者功能不足造成的自身免疫性疾病。通过观察健康人及糖尿病病人外周血调节T细胞 (CD4+CD25+与CD4+CD25+FOXP3+) 数量的差异及其与血清胰淀素水平的相关性，并使用自发T1DM模型NOD小鼠及大样本T2DM尸体解剖材料，观察hA对胰岛的免疫功能的影响。我们发现hA对胰岛具有免疫保护功能，该功能可能是通过强化调节性T细胞的功能而实现的。主要发现如下：(1) 与健康人相比，T1DM病人外周血CD4+CD25+与CD4+CD25+FOXP3+两类调节T细胞数量显著减少(p<0.05)，血清中炎症因子TNF-和IL-6显著上升。T2DM病人（n=5642）与健康人（n=7378）相比，CD4+CD25+FOXP3+细胞也显著性较少（p<0.001），炎症因子TNF-beta和TGF-alpha显著上升。说明调节性T细胞对糖尿病病的发生发展具有重要作用。(2) T1DM病人和T2DM外周血清胰淀素水平与淋巴细胞数量及白细胞数量呈负相关，提示该作用可能与血清胰淀素水平有关。（3）hA可以诱导人外周血单个核细胞（PBMC）转化为CD4+CD25+Foxp3+调节性T 细胞。（4）NOD小鼠在注射hA后，胰岛中表达的胰岛素显著性增加。同时我们也观察到hA可以刺激NOD小鼠脾脏TGF-beta和TLR-4的表达。与对照组相比，hA组NOD小鼠TGF-beta的表达了增加了50.5% （p=0.002），并且小鼠脾脏内的Treg显著性上升（3.27±0.44% vs. 1.78±0.31% ；p<0.05）。提示人类胰淀素hA可以防治NOD小鼠自发性自身免疫性T1DM，保护胰岛细胞避免自身免疫损伤。（5）通过对观察人体胰腺组织在正常及病理状态下的改变，证实了hA以及hA形成的淀粉样沉积物可能保护炎症损伤。(6) 我们在花生、瓜果及蔬菜中成功的培植了表达有hA的植物，进一步动物试验验证了表达有hA的蔬菜瓜果确实具有改善血糖与自身免疫的作用，该转化成果获得了国家发明专利的授权，说明了hA对于糖尿病、自身免疫性疾病的预防及治疗具有潜在的临床应用价值及前景。