促进胰岛微环境重建和再血管化的功能纳米材料的构建及其在胰岛移植中的应用研究

The disruption of islet extracellular matrix and inefficient revascularization decrease islet viability, leading to immediate loss of islet graft function and the limited therapeutical effect of islet transplantation. The previous studies have suggested the usefulness of self-assembling peptide to enhance the islet viability and function. If the functionalized self-assembling peptides which are able to reestablish islet microenvironment as well as to promote angiogenesis can be designed, their applications would significantly improve the therapeutical effect of islet transplantation..In this study, we plan to design several functionalized self-assembling peptides which are combined with amino acid sequences binding to the islet integrins and delivery of VEGF, and to character their morphology and physicochemical properties. Then, a three-dimensional islet culture model can be established by using peptide hydrogels in vitro, and the effects of functionalized peptides on improving the viability and insulin-secreting of rat islets is evaluated under normal/hypoxic condition. The favorable functionalized peptides are selected for in vivo study. The peptides and rat islets are co-transplanted into the diabetic nude mice under the renal capsule; the function, apoptosis, viability and revascularization of grafts are analyzed after transplantation. The feasibility of enhanced islet viability and function by using functionalized peptides is also determined. The changes of signaling pathways which involved in the regulation of islet viability and function are investigated to explore the interaction between functionalized peptide and islet, and its molecular mechanism. If the purpose of our study is achieved, it may not only increase the rate of successful transplantation, but also prolong the survival time of islet graft, and may provide a novel strategy for clinical islet transplantation.

胰岛细胞外基质损伤和再血管化障碍是造成胰岛早期大量失功，制约胰岛移植疗效的重要因素。前期研究已表明自组装多肽具有增强胰岛活性和功能的作用，若能在此基础上构建同时促进微环境重建和血管生成的功能纳米材料，有可能显著改善胰岛移植效果。.本项目拟：1）构建一系列含有不同的胰岛整合素识别序列和负载VEGF的功能化自组装多肽材料，检测其微观结构及理化性质；2）采用多肽水凝胶建立三维培养模型研究常规/缺氧条件下材料对大鼠胰岛存活和胰岛素分泌的影响，筛选能有效增强胰岛功能的材料；3）将材料与大鼠胰岛共移植到糖尿病裸鼠肾包膜下，监测移植后胰岛功能变化，分析移植物存活、凋亡及再血管化情况，评价材料增强胰岛活性功能的可行性；4）研究调控胰岛活性和功能的关键信号通路变化，阐明材料与胰岛相互作用的分子机制。若获成功不仅有望提高胰岛移植成功率，而且能延长胰岛的有效存活时间，为临床胰岛移植研究提供新的思路。

胰岛移植是治疗 1 型糖尿病的有效方法，但移植后早期β细胞大量凋亡和失功是降低移植疗效，制约其临床应用主要障碍。造成β细胞损伤的诱因错综复杂，其中β 细胞的细胞外基质（ECM）损伤、缺血缺氧和炎症损伤是重要因素。因此，在本项目针对上述引起β 细胞丢失的关键因素展开研究，通过开发功能化纳米材料降低β 细胞损伤，改善其移植后存活情况。首先，我们针对β 细胞的ECM损伤设计了一种模拟天然胰岛ECM组分-四型胶原和纤维连接蛋白的功能化自组装肽KLD-F，其可自组装成纳米纤维构成的水凝胶。采用KLD-F凝胶三维培养大鼠INS-1 β细胞可形成胰岛样细胞团，并促进β细胞的ECM蛋白分泌、细胞外微环境重建和细胞-细胞间相互作用，增强β细胞的增殖和胰岛素分泌能力，其作用机制与β细胞的整合素α5/β1-FAK/ERK- cyclin D1/p27信号通路激活有关。该结果提示，细胞外微环境对维持β细胞的正常活性和功能具有重要作用，采用模拟ECM的自组装肽促进β细胞微环境重建是一种改善β细胞功能，提升胰岛移植疗效的潜在方法。针对β细胞缺氧及炎症损伤，我们设计了阳离子自组装肽/肝素系统缓释具有抗炎及促血管生成双重作用的肝细胞生长因子（HGF）。阳离子自组装肽KLD2R能自组装形成具有三维纳米纤维结构的水凝胶，对肝素具有较强的结合和固定能力。KLD2R/肝素凝胶能显著延长HGF缓释时间，降低缺氧及炎症诱导的β细胞凋亡，维持其胰岛素分泌能力，其作用机制与抑制NFκB/p38信号通路，减少炎症因子表达及淋巴细胞浸润有关。KLD2R/肝素-HGF凝胶还能促进体外内皮细胞和体内β细胞移植物的血管生成。该研究表明，阳离子自组装肽通过静电作用吸附肝素，增强对HGF的结合和缓释能力，延长HGF的抗炎及促血管生成作用，降低缺氧及炎症等因素造成的β 细胞损伤。此外，我们还采用代谢组学方法对缺氧诱导的β细胞损伤模型进行了研究。试验结果显示，β细胞对缺氧损伤非常敏感，随缺氧时间延长其活性显著降低。缺氧组与正常组β细胞间存在明显的代谢差异，其中涉及柠檬酸循环、线粒体电子传递链、糖异生/糖酵解和酮体代谢等代谢紊乱。该结果提示，线粒体功能受损是β细胞缺氧损伤的首要代谢改变，靶向受损线粒体设计功能化纳米材料递送活性药物是保护β细胞缺氧损伤的潜在途径。