祛瘀生肌法通过Follistatin/Activin信号通路改善糖尿病溃疡炎症微环境的相关机制研究

Chronic inflammation is one of the main causes of the refractory healing in diabetic ulcer. Inflammation microenvironment, which influences the proliferation and immigration of cells, has been a focus of the recent research. Based on the final reports of the previous Youth Fund, which discovers that: 1)Although some TGFβ is negative around the diabetic ulcer wound in some clinical “deficiency mixed up with stasis” syndrome patients, its downstream related inflammatory factors including pSmad2、NF-κB is positive. 2) Gene chip database shows that Chinese medicine which removes stasis and promotes granulation can up-regulate Follistatin protein around the diabetic ulcer wound and the gene expression which is related to cell immigration and adhesion will be up regulated. This application centers on the scientific hypothesis that the inflammation microenvironment of the diabetic ulcer is ameliorated via the Follistatin/Activin/Smads signaling pathway. We will use db/db diabetic ulcer mice model platform, exogenous recombinant human protein, in situ hybridization, immunofluorescence and specific gene knockout/transgen mouse keratinocyte cultured in vitro to study the time-series intervention of Chinese medicine which removes stasis and promotes granulation on the Follistatin/Activin and its downstream target genes during the process of wound healing. The purpose of this study is to enrich the scientific connotation that removing stasis is beneficial to promoting granulation and promoted granulation won’t lead to hypertrophic scar.

慢性炎症是糖尿病皮肤溃疡持续难愈的重要原因之一，炎症微环境影响细胞的增殖与迁移。本研究基于前期发现：1）尽管部分“虚瘀夹杂”证候糖尿病溃疡创面TGFβ阴性表达，但其下游炎症相关因子（pSmad2、NF-κB）却为阳性表达。2）基因芯片数据库显示祛瘀生肌中药上调糖尿病溃疡创面Follistatin蛋白，且与细胞迁移功能相关的基因表达亦增强。本申请围绕祛瘀生肌中药可能“通过Follistatin/Activin/Smads信号通路改善糖尿病溃疡炎症微环境”的科学假说，利用db/db糖尿病小鼠溃疡模型平台，综合运用重组人源蛋白、原位杂交、流式细胞检测和免疫荧光技术，以及特定基因敲除、转基因小鼠细胞体外培养等方法，通过深入研究祛瘀生肌中药对Follistatin/Activin及其下游靶基因在创伤愈合过程中的时序干预，丰富“祛瘀利于生肌，生肌不致成瘢”学术观点的科学内涵，有利推动该法在临床的运用。

慢性炎症是糖尿病皮肤溃疡持续难愈的重要原因之一，炎症微环境影响细胞的增殖与迁移。本研究基于前期发现：1）尽管部分“虚瘀夹杂”证候糖尿病溃疡创面TGFβ阴性表达，但其下游炎症相关因子（pSmad2、NF-κB）却为阳性表达。2）基因芯片数据库显示祛瘀生肌中药上调糖尿病溃疡创面Follistatin蛋白，且与细胞迁移功能相关的基因表达亦增强。因此，通过本项目研究，我们围绕祛瘀生肌中药“通过Follistatin/Activin/Smads信号通路改善糖尿病溃疡炎症微环境”的科学假说，利用db/db糖尿病小鼠，转基因小鼠溃疡模型平台，综合运用重组人源蛋白和免疫荧光技术，以及细胞体外培养等方法，阐明了以下问题：1）糖尿病溃疡与正常皮肤溃疡相比较，存在Follistatin/Activin表达的失衡，而生肌化瘀方对Follistatin/Activin存在调控作用，并可以影响下游炎症信号通路上靶基因蛋白的下调，从而促进创面愈合；2）Smad7蛋白具有拮抗TGF-beta所诱导的炎症过度表达，可以通过抑制炎症反应促进创面愈合，外源性使用人源化Smad7蛋白是临床潜在可行的有效方案。祛瘀生肌中药具有促进内源性Smad7蛋白表达的优势，该方的多物质可能是多靶点效应的生物学物质基础。以上研究从根本上回答在临床不同症候溃疡，炎症微环境不一样，所以临床表现不一样，生肌化瘀方对经典和非经典TGFβ信号通路均有调控得以阐释；3）基于转录谱分析生肌化瘀方干预糖尿病创面样本，构建了潜在的相关circRNA-miRNA-mRNA网络，并对部分进行了验证，为更好的全面深入揭示生肌化瘀方治疗糖尿病创面的分子机制奠定了坚实基础。项目研究对于丰富“祛瘀利于生肌，生肌不致成瘢”学术观点的科学内涵具有积极意义，有利推动该法在临床的运用。

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