IL-22调控角质形成细胞CCL27/CCL28及Th22细胞CCR10在银屑病发病中的作用研究

Psoriasis is a common skin disease with unclear etiology that involves dysregulated interplay between immune cells and keratinocytes. Cutaneous T cell-attracting chemokine (CTACK/CCL27) and mucosae-associated epithelial chemokine (MEC)/CCL28, that promotes migration of lymphocytes into the skin. The role of CCL27,CCL28 and CCR10 in pathogenesis of psoriasis has recently been documented but no data are available at the present time on the related mechanism in keratinocyte hyperproliferation in psoriasis. Interleukine-22 (IL-22) plays an important role in keratinocyte hyperproliferation in psoriasis, as demonstrated by our previous studies. In this study, we will use lesional skin samples and peripheral blood collected from psoriatic patients, cultured keratinocytes, cultured Th22 cells, cultured skin 3D model and a mouse model as our experimental systems. Cultured keratinocytes, skin 3D culture and mice will be treated with IL-22 and IL-22 receptor siRNA or with CCL27,CCL28 and CCR10 proteins. We will use ELISA to detect CCL27,CCL28 and CCR10 in the peripheral blood of psoriatic patients, use real time quantitative RT-PCR to detect CCL27, CCL28 and CCR10 mRNA, use immunohistochemistry and Western-blot to detect CCL27,CCL28 and CCR10 protein. We will also observe epidermal changes in our cultured skin 3D model and mouse model after treatment. We will analyze the relationship between the expression levels of CCL27,CCL28 and CCR10 and severity of psoriasis. The objective of our study is to investigate the role and mechanisms of CCL27,CCL28 and CCR10 functions in keratinocyte hyperproliferation and inflammmatory infiltration in psoriasis. This will help to establish the groundwork for elucidating psoriatic pathogenesis.

目前认为银屑病的发病主要为T细胞介导的以角质形成细胞（KC）为靶点的免疫应答反应。CCL27/CCL28及受体CCR10参与银屑病的发生。课题组前期研究证明IL-22在银屑病KC异常增殖中发挥作用。我们推测IL-22可能通过调控KC中CCL27/CCL28和Th22细胞上受体CCR10的表达参与银屑病的发病。本研究运用分子生物学，免疫学和细胞生物学等，以临床标本、培养的KC和Th22细胞、3D皮肤体外模型和动物模型为研究对象，检测银屑病患者CCL27/CCL28及受体CCR10的表达，检测IL-22、CCL27/CCL28干预前后KC和Th22细胞功能及信号通路的改变，检测IL-22干预前后CCL27/CCL28及受体CCR10表达及相关信号通路的改变，观察3D皮肤体外模型和银屑病动物模型表皮形态及皮肤炎症浸润的改变，意欲阐明CCL27/CCL28及受体CCR10在银屑病发病中的作用。

背景 银屑病是皮肤科中最为常见的慢性疾病之一，病因不清，难以治愈，给患者带来了沉重的经济和精神负担。许多研究发现银屑病患者体内存在多种趋化因子及其受体系统的异常表达，趋化因子与其受体结合后，趋化白细胞浸润到皮肤，进而参与银屑病的发生发展。内容 探讨白细胞介素22（IL-22）对HaCaT细胞黏膜相关上皮趋化因子（CCL27、CCL28）表达的影响；探讨MMP13在银屑病患者中的表达，并研究他扎罗汀（或阿维A）和窄谱中波紫外线（NB-UVB）对MMP13表达的影响。结果和关键数据 免疫组化法结果示：银屑病患者皮损中CCL27的表达水平明显高于正常皮肤。银屑病患者皮损处MMP13呈强阳性表达，血清中MMP13水平显著高于对照组。CCK8检测显示，IL-22作用于HaCaT细胞24h后，对细胞的增殖有明显的促进作用，且这种促进作用能被PD98059抑制，差异具有统计学意义。实时荧光定量RT-PCR检测显示，IL-22作用于HaCaT细胞，细胞中CCL27、CCL28 mRNA逐渐升高，均较对照组升高，差异有统计学意义；通路阻断剂组较50ug/L IL-22组降低，差异有统计学意义。用蛋白免疫印迹法检测到CCL27、CCL28蛋白水平变化的趋势与实时荧光定量RT-PCR检测到的mRNA的水平变化的趋势一致，且差异有统计学意义。免疫荧光检测显示，HaCaT细胞内CCL28蛋白主要表达在细胞质。与对照组表皮厚度和血清MMP13水平比较，咪喹莫特组表皮厚度明显增加，表皮中MMP13强阳性表达，血清中MMP13含量也显著增加；与咪喹莫特组比较，咪喹莫特+他扎罗汀组、咪喹莫特+NB-UVB组、咪喹莫特+NB-UVB照射+他扎罗汀组表皮厚度均变薄且表皮中MMP13表达减弱，血清中MMP13含量下降。阿维A和（或）NB-UVB照射对HaCaT的增殖和MMP13的表达都有抑制的作用，阿维A+NB-UVB的抑制作用最强，强于阿维A和（或）NB-UVB单独处理的结果。结论IL-22可剂量依赖促进HaCaT细胞增殖和CCL27、CCL28的表达，其可能通过MAPK-ERK1/2通路作用；维A酸和（或）NB-UVB照射可以使银屑病皮炎样小鼠模型和HaCaT中的MMP13表达量下降，维A酸联合NB-UVB照射对MMP13的抑制作用更强。