内皮细胞三维立体培养包被构建血管化胰岛联合骨髓间充质干细胞治疗糖尿病大鼠的研究

Our study combined endothelial cells (ECs) coating islets cultured in polyglycolic acid (PGA) scaffold and mesenchymal stem cells (MSCs) cotransplantation by intravenous infusion to decrease the graft loss by lack of blood and oxygen. ECs coating can effectively solve the problem of lack of ECs on the surface of islet. PGA scaffold provides three-dimensional cuture condition which can improve the effect of ECs coating MSCs secrete many types of active proteins and immunosuppressive factors which can improve the function of islet and proliferation of ECs, inhibit rejection and inflammatory reaction meanwhile by their effects of trophic action and immunological regulation which promote the vascularized process of islet and decrease rejection and inflammatory reaction. ECs coated islets are cocultured with MSCs in vitro to simulate the environment in vivo. The effects of ECs coating and MSCs coculture on graft function, vascularization degree an immunogenicity are studied by the methods of histochemistry, ELISA and so on. Meanwhile, the effects of ECs coating and MSCs coculture on expression of graft apoptosis and survival, proliferation and differentiation, vascular generation related molecules and stimulation degree to lymphocytes are also studied by the method of Western-blot, flow cytometry and so on. The study not only explains the mechanism of improvement of ECs coating with PGA scaffold and MSCs co-culture or co-transplantation to the survival of islet graft but also provides experimental and theoretical basis to clinical application and expansion of islet transplantation for diabetes therapy.

为减少缺血缺氧、免疫排斥及炎症反应导致的胰岛移植物丢失，本研究采用内皮细胞（ECs）体外包被胰岛后于聚乙醇酸（PGA）支架，联合骨髓间充质干细胞（MSCs）静脉注射共移植的方案。利用ECs包被解决胰岛表面ECs不足，利用PGA支架三维立体的培养环境提高ECs包被效果，利用MSCs营养支持和免疫调节作用改善胰岛功能和ECs增殖，在促进胰岛血管化同时减轻免疫排斥及炎症反应，提高胰岛移植效果。为了探讨该方法的作用机制，在体外将ECs包被的胰岛分别与PGA支架和MSCs共培养模拟体内环境，研究PGA支架对ECs包被及MSCs共培养对胰岛形态功能、血管化程度、免疫原性的影响，并研究该方案对移植物存活、增殖与凋亡、血管生成相关分子的表达以及对免疫细胞激活的影响。本研究不仅可阐明ECs包被联合MSCs共移植对胰岛移植物存活的影响及其作用机制，而且为胰岛移植治疗糖尿病的临床应用及推广提供实验基础和理论依据

移植后缺血缺氧和免疫排斥及炎症反应导致的胰岛移植物丢失的主要原因，本研究在体外将内皮细胞（ECs）包被的胰岛分别与聚乙醇酸（PGA）支架和骨髓间充质干细胞（MSCs）共培养模拟体内环境，研究PGA支架对ECs包被及MSCs共培养对胰岛形态功能、血管化程度、免疫原性的影响，同时联合骨髓间充质干细胞（MSCs）静脉注射共移植至糖尿病大鼠的方案，利用PGA支架三维立体的培养环境提高ECs包被效果，利用MSCs营养支持和免疫调节作用改善胰岛功能和ECs增殖，在促进胰岛血管化同时减轻免疫排斥及炎症反应，提高胰岛移植效果。研究结果显示，PGA支架中培养环境中，包被胰岛周围的ECs数量明显多于单纯培养组，胰岛细胞分泌胰岛素分泌量显著强于单纯培养组，PGA培养组中胰岛细胞功能、ECs包被胰岛效果显著由于单纯培养组。将两组胰岛移植至糖尿病大鼠体内，结果显示，PGA支架组大鼠血糖水平明显优于单纯移植组，移植物存活率明显高于单纯移植组，PGA支架组移植物血管生生成情况明显优于单纯移植组。PGA 支架培养的ECs 包被胰岛联合MSCs 共移植结果显示，MSCs联合PGA 支架培养的ECs 包被胰岛移植物存活时间显著延长。移植后，MSCs联合PGA组大鼠外周血CD4+、CD8+T比例以及IL-2和IFN-y水平显著降低。该方案为提高胰岛移植临床应用效果奠定了实验基础。项目资助发表SCI论文3篇。培养博士研究生2名，均已毕业取得博士学位。项目投入经费23万元，支出229355万元，各项支出基本与预算相符。剩余经费0.0645万元，剩余经费计划用于本项目研究后续支出。