新型抗菌水凝胶联合人羊膜间充质干细胞促进慢性创面愈合的作用和机制研究

Cutaneous chronic wounds seriously affect the skin to serve as a protective screen and exterior, and represent a significant burden both financially and in terms of lost quality of life for both individual patients and the health-care system as a whole. Therefore, to develop a tissue engineered skin substitute, which would enhance angiogenic responses and promote wound healing, and possesses inherent antibacterial property, has become a focus of researcher’s attention. In this proposal, a previously designed self-assembling peptide hydrogel (NP1-10) and human amniotic mesenchymal stem cells (hAMSC) was used as extracellular matrix mimicking scaffold and seed cells, respectively, to prepare an ideal antibacterial hydrogel/hAMSC composite construct which enhance angiogenesis and promote wound healing through multiple mechanisms. Firstly, we will investigate the inherent antibacterial property of the NP1-10 hydrogel and the interactions between the hydrogel and hAMSC, including the influences of hydrogel on the adhesion, survival, proliferation, differentiation and secretion of the wound healing related factors of hAMSC. Secondly, non-contact co-culture will be used to study the effect on the proliferation, migration, secretion and angiogenesis of cutaneous fibroblasts, keratinocytes and vascular endothelial cells by the hydrogel/hAMSC construct. On this basis, a chronic wound model will be established in diabetic rats, then histological, histochemical and molecular biological methods will be used to investigate the effect of hydrogel/hAMSC construct on the acceleration of cutaneous chronic wound healing, with increased re-epithelialization, cellularity, and angiogenesis, and further study the possible roles of hydrogel/hAMSC construct in chronic wound healing process.

慢性创面严重影响皮肤屏障功能和外观，给患者和医疗卫生系统带来了沉重的压力和经济负担。因此，开发一种能够多途径促进血管生成和创面愈合，同时具有抗菌活性的组织工程皮肤替代物一直是研究人员关注焦点。在本项目中，我们将在前期工作中所设计的新型自组装短肽水凝胶（NP1-10）作为细胞外基质模拟支架，将人羊膜间充干细胞（hAMSC）作为种子细胞，制备出一种能够通过多途径促进慢性创面血管再生和愈合的新型抗菌仿生复合材料。我们首先研究NP1-10水凝胶的抗菌活性及其和hAMSC之间的相互关系，包括对hAMSC粘附、存活、增殖、分化及创面修复相关因子分泌等的影响等，然后用非接触式共培养的方式研究NP1-10水凝胶/hAMSC复合材料对成纤维细胞、角化细胞和血管内皮细胞增殖、迁移、分泌和成血管能力的影响。在此基础上，构建糖尿病大鼠慢性创面模型，以组织学、免疫组化和分子生物学等方法，观察复合材料促进慢性创面愈合机理。

自组装是创造纳米材料最有效的方法之一。肽基材料由于可以模拟细胞外基质的复杂结构而在生物医学领域展现出很大的应用潜力。在我们前期工作的基础上，我们主要做了以下几点工作：(1) 一条缩写为KL的短肽可以在不同pH溶液中形成β折叠结构，并在生理条件下形成水凝胶。人羊膜间充质干细胞(hAMSCs)以及创面修复相关细胞HSF和HaCaT可以在KL凝胶中存活。在凝胶中，hAMSCs的bFGF、VEGF、PDGF-BB和TGF-β1的表达量要高于普通的2D培养组。同时KL凝胶可以同hAMSCs发挥协同效应促进HSF、HaCaT 和HUVEC的增殖，上调bFGF、VEGF、 TGF-β1、KGF、Collagen I 和Fibronectin等蛋白的分泌和表达，增强HUVEC成管能力；(2) KV凝胶也具有良好的细胞相容性。KV凝胶联合hAMSCs能进一步促进α-SMA、CD31、VEGF 和Ang-1的表达而促进糖尿病大鼠创面血管新生，并增加创面胶原沉积，促进创面细胞增殖，调节创面免疫反应。同时随着接种在KV凝胶中的hAMSCs能够通过分化参与血管和上皮的重建，从而加速创面愈合。特别是，初步试验显示KV凝胶单独即可促进创面M2巨噬细胞极化并促进愈合；(3) 模拟干细胞和细胞外基质之间相互关系是调控干细胞命运的一种方法。我们研究了不同细胞粘附序列对于hAMSCs存活、增殖、干性维持和成骨分化的影响。结果表明不同功能化的RADA16凝胶都能够有效维持hAMSCs存活、增殖以及干细胞表型特征。功能化凝胶可以上调多能性标记物的表达。在成骨分化培养基中，接种于功能化凝胶中的hAMSCs的矿物沉积和成骨分化相关基因的mRNA表达也会增强。整合素α ,β亚基的上调提示机制可能与整合素介导的细胞与凝胶间相互作用有关。