SPRY1对银屑病皮肤固有免疫的影响及机制研究

Psoriasis is a common and clinically heterogeneous group of immune-mediated chronic proliferative skin diseases caused by genetic and environmental triggers. Innate immunity, adaptive immunity and skin barrier play crucial roles in the pathogenesis of psoriasis. However, how cutaneous innate immune sensing orchestrates adaptive immune responses is not defined. Sprouty 1 (SPRY1) is a cysteine-rich protein. Our immunoflurescent data has shown that in normal skin, SPRY1 localized in the epidermal granular layer. However, in psoriatic skin, SPRY1 is decreased or lost in the epidermis. Furthermore, SPRY1 was found to be closely associated with innate immune in skin defined by RNA sequencing (RNAseq). Therefore, to further clarify the role of SPRY1 in the innate immune of psoriasis, psoriatic lesional skin and SPRY1 transgenic mouse and knockout mouse were included to investigate the role of SPRY1 in the construction of skin barrier, regulation of innate immune and cytokines and chemokines secreted by epidermal keratinocytes. We further investigated the role of SPRY1 in the chemotaxis of innate immune cells in psoriasis and the induced psoriatic phenotype by wound, infection and imiquimod. Finally, a novel target for the therapy of psoriasis may be determined.

银屑病是一种多基因遗传的、环境因素刺激诱导的免疫异常性慢性增生性皮肤病，固有免疫、适应性免疫及皮肤屏障相关基因在银屑病的发病中发挥重要的作用。银屑病中固有免疫如何影响适应性免疫应答并进而导致皮损发生的机制不明确。Sprouty 1 (SPRY1) 是一种富含半胱氨酸的蛋白质，我们的初步研究发现，在正常皮肤中，SPRY1位于表皮颗粒层；而在银屑病表皮中，SPRY1表达水平降低或缺失。而且，我们通过RNAseq发现，SPRY1与固有免疫相关分子密切相关。因此，为了进一步阐明SPRY1在银屑病皮肤固有免疫中的作用及机制，我们以银屑病皮损及SPRY1转基因和基因敲除小鼠为模型，探讨SPRY1在调节皮肤屏障功能基因、固有免疫分子、角质形成细胞分泌的细胞因子和趋化因子等方面的作用，明确SPRY1改变对固有免疫细胞趋化的影响；探索创伤、感染及咪喹莫特等诱发银屑病表型产生的新机制，发现治疗银屑病的新靶标。

本研究以免疫组化及免疫荧光明确SPRY1在正常人皮肤及角质形成细胞中的定位及表达；分离培养正常人角质形成细胞，WB及qRT-PCR法检测SPRY1与细胞分化相关性；免疫组化及免疫荧光检测银屑病、湿疹、结节性痒疹患者皮损中SPRY1的表达及定位，RNAseq、qTR-PCR、WB法明确SPRY1在银屑病患者皮损区角质形成细胞与正常人角质形成细胞中的表达差异；将LPS、Poly I:C、双氧水及炎症因子（IL-6、IL-17A、IL-23、TNFα）及银屑病治疗药物（氯倍他索、.MTX、卡泊三醇、阿维A）与体外培养的正常人及银屑病患者皮损区角质形成细胞共孵育，以WB及qRT-PCR法比较以上处理因素对SPRY1在相应角质形成细胞中表达的影响；以免疫荧光检测SPRY1与抗菌肽LL37在正常人及银屑病患者组织及银屑病患者邻近皮损区角质形成细胞中的表达与定位；构建小鼠创伤模型，通过Masson三色染色、免疫组化及WB比较SPRY1转基因小鼠与WT小鼠创伤愈合过程中的差异；以IMQ构建小鼠银屑病模型，通过皮损区HE 染色比较表皮厚度、真皮血管数量，免疫荧光及qRT-PCR比较造模后SPRY1的表达差异，qRT-PCR及ELISA方法比较炎症因子、表皮屏障、固有免疫等相关基因及蛋白的改变；以流式细胞技术检测造模后真皮、皮肤引流淋巴结及外周血中炎症细胞的改变。结果发现：1、SPRY1连续表达于人皮肤颗粒层，参与角质形成细胞晚期分化；2、在银屑病患者皮损区SPRY1表达减少或缺失，药物治疗后的皮损区SPRY1表达恢复；3、LPS、Poly I:C、H2O2及银屑病相关炎症因子抑制正常人角质形成细胞SPRY1的表达；氯倍他索、MTX、卡泊三醇及阿维A可促进银屑病皮损区角质形成细胞SPRY1的表达；4、抗菌肽LL37抑制正常人角质形成细胞中SPRY1表达；5、过表达SPRY1可促进C57BL/6小鼠皮肤中表皮角质形成细胞凋亡、抑制表皮增殖，影响表皮分化。6、表皮特异性地过表达SPRY1延迟C57BL/6小鼠皮肤创伤愈合，影响创缘肉芽组织成熟机化，抑制增生表皮的爬行；7、过表达SPRY1减轻IMQ诱导的C57BL/6小鼠银屑病样皮炎表型，影响表皮屏障、固有免疫相关基因的表达，此外还抑制了小鼠真皮中IL-17A+γδT细胞增加及moDCs的聚集。因此，SPRY1在银屑病中发挥负向调节的机制