低氧对人尿源性干细胞生物学特性的影响及其修复皮肤损伤的作用与机制研究

Urine derived stem cells (USCs), a new type of adult stem cells found in recent years, is likely to be an ideal source of seed cells for cell therapy because of their noninvasive sample collection, abundant supply and other advantages. Hypoxia, which exerts multiple effects on stem cell functions, is a common feature of stem cell niche, and stem cells grafted into injured tissues need to adapt to hypoxic microenvironment. However, the biological characteristics of USCs under hypoxia remains unknown, and whether hypoxia influences the regenerative ability USCs have not been reported yet. We have successfully isolated and characterized USCs under normoxia. Moreover, we found that hypoxia enhances the proliferation and clone formation of USCs, suggesting a beneficial effect of hypoxia on USCs expansion and their regenerative ability. The first aim of this project is to characterize the biological features of USCs under hypoxia, including but not limited to their stemness maintenance, multi-differentiation capability and paracrine effects, which will provide useful information for in vitro expansion of USCs. Furthermore, a full-thickness skin defects model is used to analysis the effect of hypoxia preconditioning on USCs regenerative ability. To explore the repair mechanism, we will determine the in vivo fate of transplanted USCs and their paracrine effects with focus on USCs exosomes. Taken together, this study will provide useful information for USCs expansion and their clinical applications for regenerative medicine.

近年发现的尿源性干细胞（USCs）具有取材无创、来源丰富等优势，可能成为理想的自体干细胞来源。低氧是体内干细胞龛的基本特征，也是干细胞移植修复损伤必须适应的微环境。低氧条件下USCs的生物学特性如何？其是否影响USCs的组织修复能力？影响的机制是什么？这些问题均未见报道。前期我们已对常氧条件下USCs生物学特性进行了研究，并发现低氧能够促进USCs增殖和克隆形成，可能成为USCs高效扩增方法并促进其组织修复能力。本项目将系统比较常氧/低氧条件下USCs干性维持、分化能力、旁分泌等生物学特性，建立高效扩增USCs的培养方案；进一步采用皮肤缺损模型观察低氧预处理对USCs修复能力的影响，从干细胞分化替代和旁分泌作用两个角度探讨可能的作用机制，着重分析USCs外泌体的损伤修复功能，并寻找关键调控分子，为USCs用于组织修复再生提供实验依据。

尿源性干细胞是新型成体干细胞，具有取材无创、来源丰富等优势。低氧是干细胞移植必须适应的损伤组织微环境。本项目主要研究低氧培养条件下USCs的基本生物学特性，探究低氧预处理是否影响USCs的创面修复能力以及可能的作用机制。我们发现USCs是异质性干细胞群，主要包含生物学性质不同的两种细胞亚群，分别呈长梭形和米粒样形态，它们的增殖能力、克隆形成能力、迁移能力和多向分化能力存在差异。采用低氧培养模型，我们发现低氧提高USCs的增殖能力和克隆形成能力，影响其成骨、成脂和成软骨分化能力，不影响其干性基因表达水平，促进其分泌促血管新生相关的生长因子。采用微载体悬浮培养方式，我们发现该培养方法能够高效扩增USCs，并且维持细胞表型不变。与胎盘和骨髓来源MSCs相比，USCs具有相似的细胞表型，但体外成管能力更强。与常氧构建方法相比，低氧预处理促进USCs的创面修复能力，其创面愈合速率、血管化、再上皮化、胶原沉积与重塑情况均更好。低氧预处理主要通过提高USCs的旁分泌作用来促进创面愈合，而不是通过分化替代机制来促进创面愈合。