钠离子通道Nax对增生性瘢痕形成的作用及其表观遗传学机制的研究

The reduced hydration condition (water loss condition) that the skin wound suffered is a major factor that enhances the local imflammation, leading to the formation of hypertrophic scar. Our previous researches showed that the expression of Nax in the epidermis observably increased under the reduced hydration condition, which was involved in the inflammatory factors over-expression, dermal fibroblasts activation and the dysfunction of microvascular endothelial cells. Our further studies suggested that the abnormal expression of SET9 and the following activation of Notch pathway was the potential mechanism that Nax promotes inflammation, thus leading to the formation of hypertrophic scar. Based on our previous studies, the present project plan to: Establishment of the in-vitro cells co-culture model (SKC-DFB, SKC-MVEC) and the in vivo rabbit ear hyperplastic scar model, expliciting the functions and mechanisms that Nax/SET9/Notch signaling pathway regulating the inflammation and scar formation by using the lentiviral vector-inducd dual regulation assay, FCM and Co-Immunoprecipitation assay; By using the second generation sequencing of ChIP_Seq, bisulfite sequencing PCR (BSP) and dual luciferase report system, to further determine the mechanisms of SET9-mediated histone methylation, by which Nax promotes the inflammatory genes expression and the hypertrophic scar formation. The meaningful results of this study not only elaborate the relationship between microenvironment of wound healing and the hypertrophic scar formation from the view of epigenetics, but also give us new ideas for the prevention and comprehensive therapy of hypertrophic scar.

创面愈合过程中所处的“低水合”（失水）环境是促进炎症反应及瘢痕增生的主要因素。我们前期研究显示，表皮Nax在“低水合”环境下高表达，且与炎症因子表达、成纤维细胞过度激活及血管内皮细胞功能异常有关。进一步实验发现，SET9的表达异常及其对Notch通路的激活，可能是Nax激活炎症反应、促进瘢痕增生的主要机制。在前期研究基础上，本项目拟建立体外皮肤细胞共培养模型及兔耳增生性瘢痕模型，采用慢病毒双相调控系统及靶向抑制剂，联合ChIP-Seq二代测序、ChIP_PCR及荧光素酶报告等实验技术，探讨Nax通过SET9介导的组蛋白甲基化调控Notch信号通路促进增生性瘢痕形成的潜在机制。 本项目的完成，不仅从表观遗传学的角度阐述创面愈合微环境与增生性瘢痕的关系，而且为增生性瘢痕的预防和治疗提供新靶点。

增生性瘢痕是一种临床上常见的病理性愈合现象，常继发于严重的烧伤、手术切口或外伤等创面，发病率高、预后差。虽然研究者对于增生性瘢痕的发生和发展做了大量研究，但由于其发病机制的多样性、复杂性和不确定性，临床上仍缺乏特别有效的治疗方法。我们前期研究发现，长期处于湿性环境的粘膜上皮的损伤，如口腔黏膜、阴道粘膜等，其创面愈合后无增生性瘢痕的形成，其处于的“湿性愈合环境”可能是促进该类“完美的愈合”的主要因素。相反，皮肤创面所处的“低水合”环境（失水环境），则可能是促进瘢痕增生的主要原因。因此，明确创面所处的“湿性环境”的变化影响并调控增生性瘢痕形成的确切机制，对于防治增生性瘢痕至关重要。因此，我们针对上述理论展开研究，发现，表皮钠离子通道Nax是皮肤表皮细是感知创面所处环境湿度变化的关键信号分子（“感受器”），在创面形成后的“低水合”环境下，Nax通过上调SET9介导的H3K4双甲基化（H3K4me2）、继而激活Notch信号通路（“传感器”），从而促进增生性瘢痕的形成。而靶向、精准干预上述“感受器”和“传感器”，是逆转“低水合”环境损伤、抑制增生性瘢痕形成的潜在有效治疗措施。

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