创面愈合不同阶段微环境对人脐带间充质干细胞外泌体组分及生物学效应的影响研究

Chronic wound (delayed wound healing) and pathological scar (wound overhealing) have been a major problem in clinical medicine. The effect of MSCs in promoting wound healing and reducing scar formation has been acknowledged. However the defect in industrialization and the potential risk of clinical transformation is its obvious deficiency. Recent studies have confirmed that MSCs exosomes have the same biological effect as MSCs. But the biological effect of MSCs depends on the microenvironment of transplant site to a great extent, which suggests that the traditional method of extracting exosomes from MSCs culture used at present might be scentifically problematic. In view of the obvious differences between the biological effects of MSCs in the inflammatory stage and the moulding stage of the wound, the MSCs exosomes was extracted under the condition of simulating the wound inflammation and shaping phase in vitro. In vitro and in vivo, fibroblasts and macrophages were used as target cells to study the difference of components and biological effects between exosome extracted from MSCs cultured in the simulating inflammatory stage and the moulding stage of the wound in vitro and universally cultured ones. To verify the validity of the hypothesis that the MSCs exosome study needs to be combined with the microenvironment of the transplant site, we hope to provide a new research idea and direction for the basic and clinical transformation of the MSCs exosome.

慢性创面(创面延迟愈合)与病理性瘢痕（创面过度愈合）一直是临床医学的重大难题，MSCs促进创面愈合减轻瘢痕形成的效应已被证实，MSCs在产业化上存在的缺陷及临床转化潜在的风险是其明显不足。新近研究证实MSCs外泌体具有与MSCs等效生物效应，又规避了MSCs的不足与风险，是目前再生医学的前沿方向。但MSCs生物学效应在很大程度上取决于移植部位的微环境，提示目前以传统培养MSCs提取外泌体的方法可能存在明显科学问题。鉴于MSCs在创面炎症期和塑形期的生物效应存在明显差异，本项目在体外模拟创面炎症期及塑形期微环境条件下提取MSCs外泌体，与传统培养MSCs提取的外泌体在体内外以成纤维细胞和巨噬细胞为靶细胞进行对比研究，探讨其组分及生物效应上的差异，验证我们提出的MSCs外泌体研究需要结合移植部位微环境的假设是否成立，希望能够为MSCs外泌体的基础及临床转化研究提供一个新的研究思路与方向。

间充质干细胞（MSCs）来源的外泌体（MSC-Exos）在皮肤修复和再生方面显示出了良好的结果。然而，与传统实验室条件下培养的MSC-Exos不同的是，来自某些疾病微环境预刺激的MSC-Exos的治疗效果尚不清楚。为了评估炎症性皮肤伤口或瘢痕微环境预刺激的MSC-Exos对皮肤修复和再生的影响，我们用含有炎症因子（TNF-α和IL-1β）的炎症性皮肤伤口或瘢痕组织提取物刺激人脐带间充质干细胞（hUC-MSCs）。我们提取了hUC-MSC-Exos，通过透射电子显微镜（TEM）、纳米粒径和免疫印迹进行鉴定。采用全皮肤缺损创面模型和兔耳瘢痕模型，评价不同条件下获取的Exos对皮肤修复和再生的影响。含有炎症因子（TNF-α和IL-1β）的炎症创面组织提取物刺激获得的hUC-MSC-Exos（IF+ISH-Exos）加速早期创面愈合，增强胶原沉积，对伤口愈合早期炎症水平的调节作用。含有炎症因子（TNF-α和IL-1β）的瘢痕创面组织提取物刺激获得的hUC-MSC-Exos（IF+STH-Exos）在预防和治疗瘢痕形成方面具有显著的优势，如抑制塑料期瘢痕的形成，改善瘢痕的组织结构，调节瘢痕的炎症水平。在体外，IF+ISH-Exos促进了成纤维细胞的迁移、增殖和激活，而不影响肌成纤维细胞的功能调节。IF+STH-Exos可抑制肌成纤维细胞的迁移、增殖和激活，但在成纤维细胞的功能调节方面没有优势。我们的研究表明，用模拟移植部位的局部微环境对MSCs进行预处理所获得的外泌体可以在伤口愈合的各个阶段发挥更“精确”的治疗作用。