脱细胞支架再造胰岛素分泌器官的基础研究

Recent studies have demonstrated that the three-dimensional (3D) cell culture can provide an ideal cubic environment for cell growth. Furthermore, the 3D cell culture system established on whole organ acellular scaffold via bioreactor offers an ideal skeleton of extracellular matrix and supplies nutrients from its circulation system, which are more favorable for cell growth and differentiation whatever in the aspects of physiology, biochemistry, geometry and space. Previously, we made beta like cells by transfecting bone marrow mesenchymal stem cells and iPS cells with PDX-1, MafA and NeuroD, the crucial genes in beta cell development, and identified that they had a stable insulin-secreting function by in vitro and in vivo experiments. Moreover, we also made some whole organ decellularized scaffolds. Based on these results, we intend to complete the following tasks. First, to establish a 3D culture system favorable for beta like cell growth and differentiation. Second, to seed beta like cells and endothelial progenitor cells to this system and regenerate an artifical organ with a complete circulation system and insulin secretion function, and to implant this organ to diabetic animals and examine its physiological function. Last, to study the effect of decellularized scaffold and endothelial progenitor cells on beta like cell growth, differentiation and functioning and explore the underlying mechanism. All of our work will provide the evidence for the therapy of diabetes via the transplantation of the regenerated insulin-secreting organ.

研究发现，三维（3D）细胞培养能提供理想的立体环境，更有利于细胞的生长。利用全器官制备的脱细胞生物支架结合生物反应器所构建的3D培养体系既能提供理想的细胞外基质构架，又能模拟循环系统动态供给培养液，在生理生化和几何空间等方面更有利于细胞的生长分化。我们先前将β细胞发育的关键基因PDX-1、MafA和NeuroD导入骨髓间充质干细胞和iPS细胞，成功分化为类β细胞，经体内外鉴定其分泌胰岛素功能稳定，同时还成功制备了多种脏器的脱细胞支架。在此基础上，本课题拟完成：1、构建适宜类β细胞生长分化的3D培养体系；2、向该体系内种植类β细胞及血管内皮祖细胞等种子细胞，再造出有血管结构、能分泌胰岛素的器官，并移植到糖尿病动物模型体内，监测其生理功能；3、运用生物分析方法，探索脱细胞生物支架3D培养体系和内皮祖细胞对类β细胞生长分化及生理功能发挥的作用机制。为再造胰岛素分泌器官移植治疗糖尿病提供理论依据。

糖尿病是以血糖升高为特征的严重危害人类健康的全球性疾病。其最佳治疗方法是胰岛或胰腺移植，然而供体组织来源短缺远不能满足庞大的患者群体的临床需求，大规模扩增的种子细胞来源进一步限制了其应用。脱细胞支架技术可为构建具有长期稳定血糖调控功能的组织工程胰腺提供理想的支架来源，同时胰岛分离技术的提高及WJ-MSCs、iPSC的发展较好的解决了种子细胞来源问题。我们拟通过优化肝脏、胰腺脱细胞制备方法；进一步拓宽理想的、具备规模化扩增的再种植种子细胞来源，阐明诱导分化作用机制；提高支架脉管系统再内皮化效率，促进血管化形成；最终成功构建具有胰岛素分泌功能的胰腺类器官。在本项目的资助下，首先通过门静脉灌注1% Triton X-100/0.1%氨水、0.9%生理盐水，成功获得超微结构完整，且原有ECM成分和活性因子较大程度保留的肝脏和胰腺脱细胞支架，为再种植细胞定植、生长、增殖等功能发挥提供理想微环境；进一步采用三维动态循环灌注培养法经门静脉一次或分次注射，再种植MIN-6、胰岛细胞团簇、经WJ-MSCs和iPSC诱导分化获得的胰岛素分泌细胞，发挥短期血糖调控功能；初步阐明miRNA-690 通过靶向 Sox9 /Wnt/β-Catenin 通路调控小鼠 iPS 细胞分化为胰岛素分泌细胞，长链非编码 RNA Gm10451 作为 miRNA-338 的竞争性内源 RNA 调控 PTIP 促进胰岛类 β 细胞体外分化；为提高再种植细胞氧气和营养成分的供给，促进长期稳定血糖调控功能，进一步通过内皮祖细胞种植、肝素化修饰支架后再种植脐静脉内皮细胞、成纤维细胞与内皮细胞共培养等方法促进支架脉管系统再血管化，有效的提高了内皮化覆盖率，体内移植结果表明，能够进一步提高再种植种子细胞功能的发挥。本研究有望为复杂器官的构建提供理论与实践指导，并为进一步完善胰腺类器官的构建及临床前研究提供有效的技术支撑。