基于直接重编程策略的自体细胞替代治疗促进糖尿病创面愈合作用及其机制的研究

iPSC has become a new breakthrough in the area of stem cell research and led regenerative medicine into a new era. It has huge potential in cell therapy but is hampered in clinical use by its high oncogenicity, low efficiency and time-consuming procedures. Direct reprogramming strategy overcomes these obticles of iPS technology faced in previous studies. This innovative strategy holds great promise for the application of iPSC-based cell therapy in clinical use and broadens the perspectives of clinical autologous cell-replacement therapies based on its potential to make direct transdifferentiation occur from a type of cells into another type without establishing pluripotent state first. However, the process and mechanisms of this direct conversion between different cells is not fully understood, and clinical potency of the strategy is also not elucidated. In our study, the primary keratinocytes from diabetic mouse isolated by non-invasive method are transdifferentiated directly into endothelial progenitor cells by using direct reprogramming strategy in vitro. These induced EPCs are then applied to athymic mouse model of diabetes wound to promote angiogenesis and nerve regeneration in wound. Consequently, the process of wound healing in experimental group are accelerated. In this study, we explore the process and mechanisms of direct transdifferentiation from keratinocytes to endothelial progenitor cells and target angiogenesis of diabetes wound for the purposes of cell therapy. The combination of direct reprogramming strategy and autologous cell replacement therapy will provide a theoretic and experimental basis for optimal utilising cells in diabetes wound repair and help us to develop a new method of cell therapy for diabetes wound with high specificity, sensitivity, safety and efficacy.

诱导多能干细胞（iPSC）为干细胞领域的新突破，引领再生修复医学进入新时代，在细胞治疗中具有巨大潜力，但其高致瘤性、低效率、耗时久成为临床应用的障碍。直接重编程策略克服了iPSC的应用障碍，将一种终末分化的细胞直接转分化为另一种细胞而无需先建立细胞全能性。这一新策略使得以iPSC为基础的细胞治疗应用于临床成为可能，开拓了临床应用前景，而其过程和机制以及临床应用潜能尚未阐明。本研究拟使用无损伤方法获得糖尿病小鼠角质形成细胞，采用直接重编程策略体外直接转分化为内皮祖细胞，并应用于糖尿病创面动物模型，促进创面血管新生及神经再生，加速创面愈合。课题组在探讨角质形成细胞向内皮祖细胞直接转分化过程及机制的基础上，以糖尿病创面血管新生为治疗靶点，将直接重编程策略的优势与自体细胞替代治疗有机结合，为找到糖尿病创面修复的理想细胞奠定理论基础和实验依据，为糖尿病创面特异敏感、安全高效的新型细胞治疗开辟新思路。

糖尿病患者皮肤组织中血管病变，创面中新生血管生成迟滞，肉芽组织形成障碍，可能是糖尿病创面难愈的重要原因。作为影响创面新生血管形成的重要细胞，内皮祖细胞（Endothelial progenitor cells, EPC）在创面愈合过程中扮演重要的角色。本项目拟通过重编程技术诱导小鼠毛囊角质形成细胞转分化为EPC，提高创面内EPC的数量，从而达到促进糖尿病创面愈合的目的。我们的实验发现，在重编程的诱导转分化阶段，单纯在培养基中加入VEGF、EGF、bFGF 等生长因子不能成功诱导小鼠毛囊角质形成细胞转分化为EPC。为探索更适合的转分化诱导培养基，结合干细胞的旁分泌特点，我们尝试检测人脐血内皮克隆形成细胞的条件培养基（Endothelial colony⁃forming cells conditioned medium, ECFCs-CM）内所含的细胞因子成分，以及其对人真皮成纤维细胞、人脐静脉血管内皮细胞生物功能的影响。抗体芯片检测发现，ECFCs-CM中含有34中强阳性表达的细胞因子，如PDGF-BB、EGF、bFGF。细胞实验表明，ECFCs-CM可显著促进人真皮成纤维细胞和血管内皮细胞的增殖、迁移能力，同时也能显著提高血管内皮细胞的成管能力。动物实验则进一步表明，ECFCs-CM具有促进糖尿病小鼠创面愈合的能力。结合生物信息学分析，我们还发现糖尿病创面内micRNA与mRNA差异表达的基因，为下一步机制研究提供实验基础。研究结果将为成功实现角质形成细胞直接重编程为血管内皮祖细胞提供前期工作准备。