MicroRNA-21激活干扰素通路促进红斑狼疮皮损形成的分子机制及羟氯喹的逆转作用研究

Cutaneous lupus erythematosus (CLE) is characterized by the skin inflammation caused by apoptotic keratinocytes accumulated in the epidermis. The pathological mechanism of CLE is still unknown. Type I/ III interferon (IFN) signaling pathways in keratinocytes have been confirmed to play an important role in the pathogenesis of CLE. We have previously identified a distinct microRNA expression profile in CLE lesions compared with healthy skin, and have found that miR-21 expression was significantly enhanced in LE lesions than that in healthy skin tissue. The results of in situ hybridization showed that miR-21 have increased expression in the epidermis of CLE lesions. Meanwhile, miR-21 has activated type I/ III IFN signaling pathways in keratinocytes. Therefore, we propose our hypothesis and will explore the molecular mechanisms of miR-21 to activate IFN signaling pathways in keratinocytes and promote the formation of lupus lesions. Furthermore, we have found that hydroxychloroquine could significantly down-regulate miR-21 expression in keratinocytes. Therefore we will investigate the role of miR-21 and its activated IFN signaling pathways in CLE patients with hydroxychloroquine treatment. Above studies will provide new therapeutic targets and means for lupus lesions, and provide theoretical and experimental basis for hydroxychloroquine treatment in CLE.

皮肤型红斑狼疮以积聚在表皮的角质形成细胞的损伤引发的皮肤炎症为特征，其发病机制不清楚。已有研究证实，角质形成细胞中I/III型干扰素信号通路的激活在红斑狼疮皮损发生机制中起着重要作用。我们前期应用microRNA芯片发现miR-21在红斑狼疮皮损中较健康皮肤组织显著上调，原位杂交实验显示miR-21在皮损表皮中表达升高；miR-21能够激活角质形成细胞中I/III型干扰素信号通路。我们据此提出本项目假设探究miR-21通过激活角质形成细胞内干扰素信号通路促进红斑狼疮皮损形成的具体分子机制。我们前期还发现羟氯喹能够显著下调角质形成细胞miR-21表达，因而将查明miR-21及其激活的干扰素信号通路在羟氯喹治疗中的作用。以上研究将为临床上治疗红斑狼疮皮损提供新的靶点和治疗手段，为羟氯喹治疗皮肤型红斑狼疮提供理论基础和实验依据。

浆细胞样树突状细胞（Plasmacytoid dendritic cells, pDCs）是产生I型干扰素（Type I interferons, type I IFNs）的最主要细胞。自身免疫性皮肤型疾病如皮肤型红斑狼疮（Cutaneous lupus erythematosus, CLE）中皮肤是pDCs浸润的主要靶点，在CLE的皮损的真表皮交界处出现大量的pDCs浸润，pDCs产生异常升高的I型干扰素在CLE病机中扮演着非常重要的作用。目前还未有研究探讨炎性条件下角质形成细胞分泌的外泌体对于pDCs的免疫调节作用。本研究选取4周龄的C57小鼠，分离并培养皮肤原代角质形成细胞，采用外源性免疫核酸刺激物poly(I:C)处理原代角质形成细胞，并对外泌体进行提取和鉴定；选取6-8周龄的C57小鼠，抽提骨髓总细胞并采用pDCs分选磁珠分选得到小鼠原代pDCs，用绿色荧光试剂PKH67标记外泌体，再与pDCs共孵育，免疫荧光技术（Immunofluorescence，IF）观察pDCs内的绿色荧光信号。荧光原位杂交技术（Fluorescence in situ hybridization, Fish）示踪外泌体内miR-21-5p；收集共孵育后的pDCs的细胞培养上清，采用酶联免疫吸附技术（enzyme linked immunosorbent assay, ELISA）检测pDCs产生干扰素-α（IFN-α），干扰素-β（IFN-β），肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的量；荧光原位杂交联合免疫荧光技术观察外泌体miR-21-5p与TLR7和TLR8的共定位情况。发现鼠源角质形成细胞来源的外泌体能够促进浆细胞样树突状细胞产生I型干扰素（IFN-α和IFN-β）及促炎性细胞因子（IL-6和TNF-α）；poly(I:C)能够显著上调角质形成细胞分泌的外泌体中miR-21-5p的表达；角质形成细胞分泌的外泌体miR-21-5p可作为pDCs内TLR7和TLR8的内源性配体，外泌体miR-21-5p被TLR7和TLR8识别后，参与调控pDCs产生I型干扰素以及促炎性细胞因子。