模拟创面生态微环境构建富含活性因子天然微型真皮支架的实验研究

Extracellular matrix-growth fator interactions play a pivotal role as “soil-nutrition” in regulating tissue regeneration. The change of chronic wounds healing involves disturbing expression of growth factors, bad reconstruction of extracellular matrix and the interrupt of its synergistic effect. Therefore, the effect of a single growth factor which is adopted at present is not perfect.Our pre-research demonstrated that the natural strcture and extracellular matrix of amniotic membrane can provide an ideal “soil” microenvironment for proliferation of fibroblasts and epidermal cells, and the Amnion-Derived Cellular Cytokine Solution (ACCS) have the same type and concertation of the cytokines as physiological wound healing, which could supply rich “nutrition” to promote healing of the chronic wounds. Based on it, we preliminary prepare amniotic membrane microparticles with three-dimensional structure, which not only preserve the intact structure of amnion,but also can self-adsorb various growth factors from ACCS with its unique mesh structure. The project intends to further adopt low-temperature plasma technology to modify amniotic membrane microparticles,fully loading cytokines from ACCS to prepare the micro-dermal scaffolds rich in active factors, which may mimicking the wound "soil-nutrition" microenviroment through its natural extracellular matrix components and various active factors. The present study is a further study on the basis of two previous studies that have been published on journals of Science Citation Index(SCI). The result expected to provide a new way for the treatment of chronic wounds.

细胞外基质-生长因子的交互协同在组织修复过程中发挥“土壤-营养”的基础作用，慢性创面愈合改变涉及细胞外基质重建不良、生长因子表达紊乱及其协同作用异常，因此目前采用单一生长因子治疗效果有限。前期研究发现羊膜天然组织结构及细胞外基质为表皮细胞、成纤维细胞粘附、增殖提供理想的“土壤”，同时羊膜来源细胞因子溶液具有和正常创面愈合相似的因子种类及浓度梯度，可为创面愈合提供丰富的“营养”。在此基础上初步研制了具有三维立体结构的微粒羊膜，其不仅完整保留了羊膜基本结构，同时其特有的网孔状结构可自主吸附羊膜来源活性因子。本项目拟进一步采用低温等离子技术表面活化微粒羊膜，充分加载羊膜来源活性因子，制备富含多种因子的微型支架，以期通过羊膜天然的细胞外基质成分及多种活性因子模拟近似生理的“土壤-营养”微环境改善慢性创面愈合。这是在发表2篇SCI论文基础上的延续和深入，该研究有望为慢性创面治疗提供新的方法和途径。

慢性创面愈合改变涉及细胞外基质重建不良、生长因子表达紊乱及其协同作用异常，因此目前采用单一生长因子治疗效果有限。前期研究发现羊膜天然组织结构及细胞外基质为表皮细胞、成纤维细胞粘附、增殖提供理想的“土壤”，同时羊膜来源细胞因子溶液具有和正常创面愈合相似的因子种类及浓度梯度，可为创面愈合提供丰富的“营养”。基于此，本项目研制了具有三维立体结构的微粒羊膜，其大小为300-600μm，其不仅去除了免疫抗原成分，而且其完整保留了羊膜基本结构及细胞外基质成分，在此基础上，结合三维旋转培养体系（RCCS）构建了含成纤维细胞的活性微型真皮支架，检测发现其具备较高活性，可分泌多种细胞因子，包括bFGF、VEGF、PDGF、HGF、NGF、TGF-β1、IL-8、IL-6等，可增强角质形成细胞和成纤维细胞的增殖和迁移效果，并能通过促进创面血管化、加快上皮化改善糖尿病小鼠创面愈合。在上述研究的基础上，本研究进一步探索制备了保存羊膜原有上皮细胞的活性羊膜微粒，并通过移植糖尿病小鼠全层皮肤缺损模型发现，其可以通过分泌释放多种活性因子促进血管化（增强内皮细胞迁移、趋化募集CD34+祖细胞）、调控创面炎症反应（调节巨噬细胞类型转变）、加快上皮化（促进表皮细胞迁移）及重塑真皮支架等多条途径改善慢性创面的愈合。本研究显示含成纤维细胞的活性微型支架和保存羊膜原有上皮细胞的活性羊膜微粒作为活性真皮支架可能为改善慢性创面愈合提供一种新的治疗方法。本研究成果发表SCI收录论文5篇，获得国家发明专利授权1项。