探索表皮分化异常对硬皮病发生及发展的影响

Scleroderma (SSc) is a connective tissue disease with unknown etiology. The pathology hallmark includes overproduction of extracellular matrix, vasculopathy and autoimmunity. Currently, there are increasing studies observing abnormalities in SSc epidermis. Genome wide association study（GWAS）from our previous study reveals that multiple SNPs in the Epidermal Differentiation Complex（EDC）region were associated with SSc, indicating that terminal differentiation of epidermis may play functional roles in SSc. In addition, keratinocyte differentiation defects were also observed in our SSc sections. Given all these, this proposal is trying to explore the functional relevance of epidermis in the development of SSc. We will start from clinical observation; systematically summarize the epidermal changes in different SSc stages; stimulate the SSc epidermis with environmental toxins and pathogens ex vivo to explore the consequence of varied epidermal barrier function in SSc. By using 2D and 3D culture of SSc-derived primary keratinocyte, we will identify whether the abnormalities of epidermal is keratinocyte cell-autonomous event. This study will reveal the functional role of epidermis in the development and progression of SSc from a new perspective, providing new ideas for etiology, early screening and treatment of SSc.

硬皮病（SSc）是一种病因不明的结缔组织病。病理上以成纤维细胞过度分泌胶原、血管损伤和多种抗细胞自身抗体的产生为主要特征。近年来，越来越多的研究报道除上述变化外，表皮层也存在明显异常。申请人所在课题组全基因组关联分析（GWAS）结果显示上皮分化复合物 (EDC)区域存在多个与SSc相关的位点，提示表皮终末分化可能在SSc的发病中具有潜在功能。申请人前期免疫染色亦发现SSc角质形成细胞分化异常。鉴于此，本项目将从临床观察出发，结合病史系统性研究SSc不同阶段的表皮形态及功能变化。通过环境刺激物及病原体孵育SSc离体表皮，探索SSc表皮变化的功能学意义。最后利用病人来源的原代角质形成细胞，进行体外2D和3D分化实验，以阐明SSc表皮异常是否源于角质形成细胞的自发性改变。本项研究从表皮的角度重新审视疾病的发生和发展，将为揭示SSc的病因、早期筛查及治疗提供新的思路。

硬皮病是一种病因不明的复杂结缔组织病，根据病变是否累积脏器可分为局限性硬皮病（LSc）和系统性硬皮病（SSc），皮肤病理以真皮层成纤维细胞过度分泌胶原为主要特征。近年来多项研究显示除真皮层变化外，患者表皮层也存在明显异常，但仍缺乏系统性研究。本项目募集27名LSc患者，对其皮损处和相邻/对侧非皮损处共计138个部位进行了超声检测及无创功能检测。结果显示LSc患者表皮明显增厚，表皮含水量显著降低且与表皮厚度呈负相关。博来霉素诱导的细胞模型转录组学数据提示角质形成细胞（KC）异常分泌IL-6。小鼠实验及细胞实验进一步证实IL6可促进KC增值，导致表皮失稳态性，皮肤含水量下降，进而出现干燥、瘙痒，可严重影响患者的生活质量。此外，活检切片显示SSc患者表皮中AHR表达异常。前期发现AHR信号通路可调节KC的增殖与分化，然而其具体作用机制尚未完全阐明。为此，我们采用CRISPR/Cas9技术成功构建AHR特异性敲除的人角质形成细胞系。在AHR配体苯并芘和并咔唑处理后，转录组学数据提示AHR可诱导ALDH1A3的表达。进一步细胞实验证实AHR可诱导ALDH1A3的表达并呈现明显时间和剂量依赖性。AHR可提高胞内ALDH1A3酶活性，进而维甲酸的生成。体内小鼠实验证实苯并芘局部涂抹可诱导小鼠皮肤中ALDH1A3的表达，提高全反式维甲酸（atRA）的含量，显著增加表皮厚度；而此生物学效应在AHR敲除鼠中则完全消失。因此，本项目证实AHR可通过ALDH1A3调控KC中atRA的合成，改变表皮厚度进而影响皮肤屏障功能。本项目的实施，使我们对硬皮病的病理有了更全面的认识，并发现了新的作用靶点，对治疗药物的开发具有重要意义。