腹腔来源的M2型巨噬细胞延长胰岛移植物存活的研究

A widely accepted therapy for type 1 diabetes (T1D) was islet transplantation. The use of immunosuppression post transplant reduced inflammatory response and rejection. At the same time, the potential dangerous side effects (infection or malignant tumor) of immunosuppression were currently of concerns. M2 macrophages were extremely versatile effector cells which have been shown to anti-inflammatiory and immune regulatiory functions. M2 macrophages also have been shown to promote beta-cell proliferation and vascularization. Mф from peritoneal could be modulated in vitro into M2 Mф which maintained a stable phenotype in culture for at least 2 weeks. So we established the peritoneal dialysis mice model, peritoneal Mф will be collected and modulated with IL-4/IL-13 to become M2a in vitro. M2 and fresh islets were transplanted beneath kidney capsule of diabetic mice through a PE 50 tube. The C-peptide and blood glucose levels were measured. An Intraperitoneal glucose tolerance test (IPGTT) was conducted to assess graft function. Furthermore, we utilized tumor necrosis factor a (TNFα)-induced damage to islet cells to model the inflammation of post-transplant explore whether the protective action of M2 was involved in TNFαproduction and nuclear transcription factor-κB (NFκB) activation. The mixed lymphocyte reaction were performed and the flow cytometry-based methods were used to quantify CD4+ and CD8+ T cell proliferation and activation. Peritoneal regulatory M2 macrophage might represent an attractive candidate to reduce dose of immunosuppressive drugs in a clinical trial of islet transplantation.

胰岛移植成为了彻底治疗1型糖尿病的手段。免疫抑制剂的使用降低了移植后的炎症反应和免疫排斥促进了移植物的存活，免疫抑制剂可导致机体抵抗力下降，诱发严重感染、恶性肿瘤等疾病。M2型巨噬细胞(M2)是一种具有多功能免疫调节的效应性细胞，不仅能够降低炎症反应和移植后的免疫排斥反应，还可促进移植胰岛细胞的增殖和血管化。腹腔中大量的单个核细胞能被活化为稳定的M2。因此本项目拟通过建立小鼠腹腔透析模型，收集腹腔单个核细胞，在体外培养诱导成M2，与胰岛细胞共移植到同种异基因糖尿病小鼠体内，观察M2对移植胰岛存活增殖和功能发挥的影响，通过体外胰岛炎症因子损伤模型检测TNFα-NFκB-p38MAPK通路研究M2降低炎症反应的分子机制，并且通过混合淋巴细胞反应实验研究M2通过检测淋巴细胞增殖和活化情况，进而探讨促进移植物存活的机理。为临床胰岛移植及其他器官移植后长期服用免疫抑制剂的问题提供了一定的实验基础。

背景 .M2型巨噬细胞(M2)是一种具有多功能免疫调节的效应性细胞，不仅能够降低炎症反应和移植后的免疫排斥反应。在本研究中，我们检测了腹腔来源的M2巨噬细胞对糖尿病小鼠移植的胰岛存活和功能发挥的影响，希望能够长期改善移植后糖尿病小鼠的血糖控制。.研究内容 .从C57BL/6小鼠腹膜透析液中分离巨噬细胞，体外诱导成M2型表型。建立Balb/c- C57BL/6 小鼠胰岛移植模型，并于分别于移植后0天3天和7天尾静脉注射M0/M2。移植后通过血糖和腹腔葡萄糖耐量实验（IPGTT）对移植的胰岛功能进行评价，进而对M2 的功能进行评估。在移植后的7天取各组小鼠外周血，通过ELISA分析血清中炎症因子含量。在移植后的30天取脾脏通过流式对淋巴细胞分群进行分析；取胰腺组织固定并通过免疫组化染色检测胰岛细胞的存活情况。体外通过M2型巨噬细胞与CD4+T细胞或CD4+CD25-T细胞共培养，流式检测CD4+T细胞和Treg细胞的增殖情况。 .结果及关键数据 .在体内研究中，M2的输注显著降低了糖尿病小鼠的血糖，更大程度地保存了胰岛细胞的结构，并改善了胰岛功能的发挥。 M2抑制炎症细胞因子的分泌，受体小鼠血清和移植胰岛中IL-10的水平显著升高。 M2输注的移植小鼠脾脏CD4+和CD8+ T细胞比例明显低于其他组小鼠。 在体外的研究中，M2共培养显著抑制CD4+ T细胞增殖，但是促进Treg细胞的比例。 .结论或科学意义 .腹腔M2型巨噬细胞输注可显著降低糖尿病小鼠的炎症浸润，促进移植胰岛的存活和功能的发挥。 腹腔M2型巨噬细胞表现出多种免疫调节特性，影响T淋巴细胞亚群分布、抑制CD4+ T细胞增殖和促进Treg细胞的积累。