烧伤创面修复过程中非编码RNA调节表皮细胞与成纤维细胞及其相互作用的研究

Epithelialization and dermal reconstruction are two major elements of deep burn wound repair, it has been found that good coverage on retained denatured dermis can prevent its secondary necrosis. We successfully used large sheet of split-thickness skin grafting with retained denatured dermis for treating deep second degree burns. Studies have shown that non-coding RNA (ncRNA) in the denatured dermis changes significantly, and ncRNA also exists in burn blister fluid and negative pressure suction liquid from burn wound. According to the study above, we established a scientific hypothesis that ncRNA may regulate epidermal cells and fibroblasts and its interaction between two kinds of cells during deep burn wound repair by certain signaling pathways. We propose to compare the difference of ncRNA expression in epidermal cells, fibroblasts and the culture medium of these cells, using denatured dermal fibroblasts in the early stage after burn and fibroblasts in granulation wound co-cultured with razor-thin graft or epidermal cells in three-dimensional environment in vitro. Combining with the time of injury repair and method of skin grafting, to thoroughly screen ncRNA associated with burn wound healing and observe their regulation on epidermal cells and heat injuried fibroblasts, and verify and evaluate them in burn blister fluid and negative pressure suction liquid from burn wound. To thoroughly study the mechanism and influence factors of deep burn wound healing, and explore the measures to improve the quality of deep burn wound healing based on ncRNA, which is of important theoretical value and wide application prospects.

上皮化和真皮重建是深度创面修复的两大内容，实践证明保留变性真皮予以良好覆盖可防其继发坏死。我科采用保留变性真皮移植整张薄皮片治疗深Ⅱ度烧伤获得成功；研究发现变性真皮的非编码RNA(ncRNA)发生明显变化，烧伤水泡液和负压抽吸液中也存在ncRNA。因此，本课题建立ncRNA可通过信号通路调控深度烧伤创面修复时表皮细胞和成纤维细胞及其相互作用的科学工作假说，拟采用表皮片或表皮细胞与变性真皮成纤维细胞或肉芽创面成纤维细胞体外三维立体共培养，比较表皮片或表皮细胞、成纤维细胞及其培养液的ncRNA表达差别；结合修复时机和植皮方式全面筛选与烧伤创面愈合相关的目标ncRNA，探讨它们对表皮细胞和热损伤成纤维细胞的调节作用及其机制，并在烧伤水泡液和负压抽吸液中临床验证和分析，深入研究深度烧伤创面愈合机理及其调控因素，基于ncRNA探讨改善深度烧伤创面愈合质量的措施，具有重要的理论价值和广阔的应用前景。

尽早修复创面是烧伤治疗的关键，ncRNA可能在创面修复过程中起重要作用。该项目通过表皮细胞与变性真皮成纤维细胞或肉芽创面成纤维细胞体外三维立体共培养，采用基因芯片比较表皮细胞对不同时期创面成纤维细胞ncRNA表达的影响，并进行生物信息学分析；同时，提取细胞培养液中外泌体并进行鉴定。.结果发现热损伤以及HaCaTs共培养可对成纤维细胞的lncRNAs、miRNAs和mRNAs表达造成影响；烧伤肉芽成纤维细胞与正常成纤维细胞存在差异表达的ncRNA；与HaCaTs细胞共培养后，肉芽成纤维细胞产生了差异表达的ncRNA；与不同的成纤维细胞共培养后，HaCaTs细胞产生了差异表达的ncRNA。这些差异表达的lncRNAs、miRNAs和mRNAs可能通过调节细胞因子-细胞因子受体相互作用、ECM受体相互作用、TGF-β信号等信号通路，调控表皮细胞和成纤维细胞的细胞周期、增殖、迁移、凋亡等生物学过程，从而影响创面愈合。生物信息学分析显示高度差异的基因之间存在lncRNAs-miRNAs-mRNAs互作用网络，LINC02206、LINC001182、LINC01783 ADAMTS9-AS2 、lncRNA BGIG9606_79960、LncRNA TSIX与lncRNA LOC105378179等可能是烧伤真皮变性及烧伤创面愈合过程中较为重要的调节因子，初步研究发现这些ncRNA可能以外泌体形式参与表皮细胞和成纤维细胞之间的相互作用，.该研究模拟临床烧伤创面修复方式，基本揭示烧伤创面ncRNA在表皮细胞和成纤维细胞及其相互作用中的变化，初步筛选与烧伤创面愈合相关的ncRNA，对深入研究深度烧伤创面愈合机理及其影响因素，以及基于ncRNA探讨改善深度烧伤创面愈合质量的措施，具有重要的理论价值和广阔的应用前景。