特应性皮炎(atopic dermatitis,AD)是一种常见且难治性的皮肤病。我们前期工作表明培土清心方疗效确切，但其治疗靶标及作用机制尚未明确。本项目将基于已发现miRNA-30a在AD患者和AD动物模型中表达显著上调，并明确其靶基因为NEDD4的研究基础上。本项目将开展运用CRISPR/Cas9技术构建miRNA-30a基因敲除小鼠，阐明培土清心方治疗AD模型小鼠疗效作用与miRNA-30a靶向NEDD4调控机制。检测并分析培土清心方对基因敲除鼠与正常鼠的皮损、Th17/Treg免疫调控等，证实培土清心方对NEDD4及其下游调控表达及疗效差异。再结合我们团队已收集的临床AD样品，采用二代测序技术检测并分析miRNA-30a与培土清心方治疗相关性进行验证。探索miRNA-30a靶向NEDD4参与培土清心方治疗AD作用及疗效机制，发掘miRNA-30a作为AD标记物提供数据支撑。

Atopic dermatitis (AD) is a common chronic refractory and relapsing disease with inflammatory skin disease. Previous studies of our research team have revealed that the traditional Chinese medicine(TCM) formula “Pei Tu Qing Xin” (PTQX) has a definite clinical effect on the treatment of atopic dermatitis. However, its therapeutic target and the specific mechanism remains unknown. This project will be based on the research that has found that miRNA-30a expression is significantly up-regulated in AD patients and AD animal models, and that its target gene is NEDD4. Our project will carry out the construction of miRNA-30a knockout mice using CRISPR/Cas9 technology, to clarify the therapeutic effect of PTQX on AD model mice and the mechanism of miRNA-30a targeting NEDD4. At the same time, we will detect and analyze the differential expression on the lesions, the immune regulation of Th17 cells/Treg and efficacy of NEDD4 and its downstream after the formula PTQX treatment in siRNA knockout and wild-type AD mice model. Then combined with the clinical AD samples collected by our team, and using second-generation sequencing technology to detect and analyze the correlation between miRNA-30a and the formula PTQX therapeutic effect. To explore the role of miRNA-30a targeting NEDD4 of PTQX in the treatment of AD and its mechanism of efficacy, and to discover miRNA-30a as an AD marker to provide data support.