MSC外泌体联合PDMS支架促进移植胰岛免疫耐受和血管再生的作用及机制

Islet transplantation is the most effective treatment for brittle diabetes. However, loss of function of the transplanted islets due to immune rejection and failure of angiogenesis remains an unsolved clinical problem. Polydimethylsiloxane (PDMS) scaffold provides a stable environment for transplanted islets by separating them from immune cells physically. Yet the angiogenesis of islets is poor. We previously found that MSCs could protect islet both in vivo and in vitro, release a large amount of exosomes, as well as inhibit proliferation of lymphocytes and promote angiogenesis, but the underlying mechanism is still unclear and remains to be investigated. In our current study, PDMS scaffold will be employed to house transplanted islets and MSC-derived exosomes will be thoroughly investigated to figure out whether this will help to induce immune tolerance and promote angiogenesis of transplanted islets. In detail, we will focus our study on investigation of: 1) the effect of MSC-derived exosomes combined with PDMS scaffold on proliferation and function of immune cells, and on apoptosis and angiogenesis of islets in PDMS scaffold; 2) analysis of the active components of exosomes by MS and microRNAs array, and unveiling the model of action of exosomes by fluorescence labeling, RNA interference, as well as targeted inhibition; detection of the activation of relative signaling pathways and its effect on downstream effectors, and 3) verification of in vivo therapeutic effect of MSC-derived exosomes combined with PDMS scaffold on islet transplantation in treatment of STZ induced diabetes rats model. In summary, we aim here to figure out whether MSC-derived exosomes combined with PDMS scaffold will help to induce immune tolerance and promote angiogenesis of transplanted islets, as well as its relative underlying molecular mechanism. The fulfillment of our current study will help to improve the clinical outcome of islet transplantation.

胰岛移植是治疗脆性糖尿病的最有效方法，免疫排斥和血管再生障碍是移植胰岛失功的主要原因。聚二甲基硅氧烷（PDMS）生物支架可提供稳定的环境，避免胰岛与免疫细胞直接接触，但影响胰岛血管生成。前期研究发现MSC能保护胰岛，MSC释放的外泌体具有促进内皮细胞血管化、抑制淋巴细胞增殖的作用，但机制不明。因此本研究拟利用MSC外泌体联合PDMS支架为移植胰岛提供定居环境，通过观察对免疫细胞增殖、功能的影响和PDMS支架内胰岛凋亡、血管生成的情况，证实外泌体联合PDMS支架的作用效果；分析外泌体的活性成分，通过靶向抑制、RNA干扰的方式，同时检测免疫细胞和胰岛内的信号通路活化情况及对下游效应分子的影响，挖掘外泌体作用的分子机制；通过治疗STZ诱导糖尿病大鼠模型，验证体内的治疗效果。初步阐明外泌体联合PDMS支架促进移植胰岛免疫耐受和血管再生的作用机制，为提高胰岛移植治疗效果提供研究基础。

胰岛移植是治疗脆性糖尿病的最有效方法，低氧、血管再生障碍和免疫排斥是移植胰岛失去功能的主要原因。本课题通过利用间充质干细胞外泌体的免疫调节、促血管生成的功能联合聚二甲基硅氧烷（PDMS）生物材料，探讨提高移植胰岛存活的途径，并深入挖掘MSC外泌体提高胰岛存活的分子机制。首先通过分离脐带MSC条件培养基中的外泌体，并进行外泌体鉴定和生物活性成分（miRNA）测序。其次，利用PDMS构建3D巨孔生物支架，联合PDMS支架、MSC外泌体、胞外基质等共同构建一个“人工胰腺”微环境，通过体外胰岛素刺激释放实验和糖尿病动物模型体内移植评价“人工胰腺”的功能和治疗效果，以及评价免疫排斥情况和血管生成情况。利用胰岛β细胞、内皮细胞体外探讨MSC外泌体对支架内β细胞低氧条件下存活、内皮细胞血管生成的影响及机制。结果显示，PDMS可成功构建稳定的三维巨孔生物支架，为移植胰岛提供稳定的物理空间。通过联合间充质干细胞外泌体，胞外基质以及装置胰岛细胞，共同构建成一个迷你的“人工胰腺”。构建的迷你“人工胰腺”能响应葡萄糖刺激分泌胰岛素的功能；将“人工胰腺”移植到糖尿病大鼠大网膜内，可降低糖尿病大鼠的血糖水平。病理结果显示，MSC外泌体可提高移植胰岛活性，减少免疫炎症反应，促进血管生成。进一步的实验结果证实间充质干细胞外泌体通过抑制低氧引起的胰岛beta细胞内质网应激反应，抑制eIF2a-chop和P38 MAPK信号通路，减少胰岛细胞的凋亡；同时可以上调Hif1a,促进胰岛内皮细胞VEGF（血管生成因子）的表达，激活mTOR信号通路，促进胰岛血管生成。促进移植胰岛存活。本研究成功利用PDMS联合间充质干细胞及胞外基质为移植胰岛构建微环境，为临床胰岛移植提供新的策略。初步阐明外泌体联合PDMS支架促进移植胰岛免疫耐受和血管再生的作用机制，为提高胰岛移植治疗效果提供研究基础。课题资助已发表SCI论文3篇，核心统计源论文3篇，申报发明专利1项。培养博士研究生1名，硕士研究生2名。