“间接谱系转化”结合纳米技术重编程小鼠胚胎成纤维细胞成为少突胶质细胞的研究

Indirect lineage conversion is an efficient method for the conversion of the differentiated somatic cells to lineage-specific progenitor cells based on the transfection with gene vectors encoding specific transcription factors, it provides a new approach for regenerative medicine. Nanoparticle as a novel non-viral vector for gene delivery, it has many advantages. The stability of nanoparticle and its functions in gene delivery and imaging tracking can be improved after being modificated on its surface. Therapeutic effect after being transplanted with stems cells or progenitor cells shows a significant decrease because some transplanted cells can’t finally differentiate into functional cells. In this project, multi-modal transfection agent (MTA) with high transfection efficiency through surface modification will be obtained. MTA/plasmid DNA (pDNA, pEGFP) complex will be prepared and the reprogramming of mouse embryonic fibroblasts (MEFs) into induced oligodendrocyte precursor cells (iOPCs) will be mediated based on the expression of transcription factors. MTA/miRNA complex will be cellular uptaked by iOPCs and up-regulate the differentiation of iOPCs into myelin-forming oligodendrocytes, which will contribute to break through the bottleneck of differentiation from OPCs into oligodendrocytes after the transplantion of stems cells or progenitor cells in the treatment of dysmyelinating disease and improve its therapeutic effect. The aim of this project was to establish a reprogramming system which can realize the functional conversion between different differentiated somatic cells by combining indirect lineage conversion and nano technology and regulating the proliferation and differentiation of progenitor cells for basic research, regenerative medicine and so on.

“间接谱系转化”技术借助基因载体，通过表达转录因子，重编程已分化体细胞成为特定谱系的祖细胞，为再生医学提供了新途径。纳米颗粒作为新型非病毒基因载体，通过表面修饰，能够增强其稳定性以及传递基因和成像追踪等的功能。研究发现干细胞或祖细胞移植时，容易出现植入细胞无法最终分化成为目的功能细胞的现象，影响其疗效。为此，本项目拟表面修饰纳米颗粒，获得功能化多模态转染试剂（MTA），构建MTA /质粒DNA复合物，通过转录因子的表达，重编程小鼠胚胎成纤维细胞成为诱导性少突胶质细胞的祖细胞（iOPCs），并借助MTA传递miRNA至iOPCs，突破髓鞘障碍性疾病治疗中植入祖细胞的分化容易卡在OPCs与少突胶质细胞之间的瓶颈，提高能够生成髓鞘的成熟少突胶质细胞的效率，改善治疗效果。建立有机结合“间接谱系转化”与纳米技术，借助对祖细胞的调控，实现已分化体细胞之间功能转换的重编程体系，用于基础研究和再生医学等。

本项目针对细胞移植治疗中存在的细胞产量少、分化控制难和安全隐患高等问题，将“间接谱系转化”与纳米技术有机结合，建立了重编程体细胞成为诱导性少突胶质细胞（iOLs）的平台。项目首先利用聚乙烯亚胺（PEI）修饰的超顺磁性氧化铁纳米颗粒（SPION）表面正电荷结合质粒DNA，通过研究该复合物的理化特性、保护作用、细胞毒性、传递机制和转染条件等，建立了高效、低毒的细胞转染体系；并利用该体系介导三种神经转录因子Olig 2、Nkx 6.2和Sox 10表达，成功地将皮肤成纤维细胞转化成为具有双向分化潜能的诱导性少突胶质细胞祖细胞（iOPCs），并通过筛选和添加细胞因子，优化了其增殖培养体系；通过在胰蛋白酶溶液中添加鸡血清和采用间隔震荡纯化方法，建立了更为高效的原代OPCs的分离、培养和纯化体系；利用PEI-SPION传递多能转录因子基因或重组蛋白，成功地将体细胞逆转化为诱导性多能干细胞（iPSCs），验证了磁性氧化铁纳米颗粒介导体细胞重编程的功效并拓展了其应用范围；通过制备细胞外基质（ECM）和解析其蛋白成分，结合条件优化，构建了3D ECM水凝胶细胞培养体系，为深入研究iOPCs和iOLs的行为和功能提供了立体平台；基于miR-219、miR-23和miR-138促进iOPCs分化成为成熟iOLs的作用，建立了利用磁性氧化铁纳米颗粒和脂质体高效传递miRNA至iOPCs的体系；通过优化药物注射的剂量和部位，建立了稳定的小鼠实验性自身免疫性脑脊髓炎（EAE）模型，改良了小鼠侧脑室细胞移植的方法，并借助芬戈莫德阻止EAE进程、改善局部微环境的作用，检测了芬戈莫德结合转染有miRNAs的iOPCs的细胞移植治疗EAE的效果，建立起了一个新型、高效、安全、快速的谱系重编程体系，借助对祖细胞的调控，实现已分化体细胞之间的功能转换，可用于基础研究和再生医学等。