基于miRNA-155调控Th细胞极化探讨清心培土法治疗特应性皮炎的疗效机制

Atopic dermatitis（AD） is a refractory chronic relapsing dermatologic disease, which is commonly seen in clinical practice. And Th cells polarization is a pivotal pathogenesis. Previous studies of our research team has revealed that purging heart fire and tonifying spleen earth tends to regulate abnormality of Th1/Th2, thus, to relieve conditions of Atopic dermatitis. However, the specific mechanism remains unknown. Besides, some other studies have indicated that expression of miRNA-155 has significantly up-regulated in AD patients resulting in target CTLA4 regulation of Th cells in proliferation and differentiation. In this setting, we assume that "Purging heart fire and tonifying spleen earth" therapeutic method might be able to restore Th1/Th2 imbalance, so as to relieve inflammation in AD patients through regulation of miR-155. Our study intends to centrifuge PBMC from treatment and control group to detect the expression of miR-155, Th1/Th2 balance and function target through Luciferase report genes and antisense oligonucleotide technology . Further more, we establish " Purging heart fire and tonifying spleen earth" intervention murine model to observe peripheral ,skin,spleen and lymph node miRNA-155 expression, Th1/Th2 cytokines, transcription factor and to testify miRNA-155 target gene proteins SHIP1,SOCS1 and CTLA4. Hence, to analyze relativity between miRNA and heart fire and spleen insufficient pattern. In this case, provide for evidence for counter-balance and molecular mechanism of treatment of "purging heart fire and tonifying spleen earth" in Atopic Dermatitis.

特应性皮炎（AD）是常见难治性慢性复发性皮肤病，其发病涉及Th细胞极化异常的复杂免疫网络。本课题组前期研究验证清心培土法疗效显著，可调节Th1/Th2平衡，但治疗靶点尚不明确。基于miR-155在AD患者中表达显著上调，靶向SHIP1、SOCS1和CTLA4可定位调节Th细胞极化的基础，我们推测通过miR-155调节Th1/Th2平衡可能是清心培土法治疗AD的疗效作用机制。本课题分离AD外周血PBMC，通过双荧光素酶报告基因和反义寡核苷酸技术探讨miR-155对Th1/Th2平衡的影响及功能靶点；以培土清心颗粒干预AD小鼠模型，运用液相芯片和实时荧光定量PCR技术，观察外周血、皮损、脾淋巴结Th1/Th2细胞因子、转录因子、miR-155与靶基因蛋白SHIP1、SOCS1和CTLA4表达；分析miR-155与心火脾虚证候比重的相关性，为阐明清心培土法治疗AD的制衡策略和分子机制提供依据。

特应性皮炎(atopic dermatitis, AD)是一种慢性易复发性、炎性皮肤病，严重影响患者生活质量，也给家庭及社会带来沉重的经济负担。是皮肤科临床上最常见的皮肤病之一。我们根据多年临床积累并结合中医药理论总结出“培土清心法”治疗AD。本研究表明，AD患者PBMC中Th1/Th2较正常组降低，且差异有统计学意义。培土清心颗粒可调控Th1/Th2细胞平衡，动物模型表明其具有较好的药理作用。同时特应性皮炎的动物模型还表明血清中miR-155与IL-4具有相关性，并且培土清心颗粒对AD样小鼠模型的脾脏细胞中的Th1/Th2及皮损miR-146a具有调控作用。表明清心培土法治疗AD可制衡Th1/Th2并干预miRNA。血清中miR-146a表达较正常组高，差异具有统计学意义（P＜0.05）。在AD患者不同证型中，心火脾虚证患者miR-146a表达水平最高，心脾积热证患者次之，脾虚蕴湿证患者再次之，血虚风燥证患者最低，但各组差异均没有统计学意义（P＞0.05）。AD患者miR-146a与SCORAD、IGA评分均没有相关性。此外，我们采用二代测序方法分析健康人群与AD患者中miRNA表达的差异，发现了特应性皮炎患者特有的miRN有6个，并且发现两组比较有统计学差异的miRNA有5个。通过对相关miRNA功能进行靶基因预测，并与特应性皮炎T细胞免疫相关的信号通路，我们探寻了miRNA功能靶基因的调控网络。同时明确了几个miRNA的靶向基因，miR-155靶向BTN2A2基因、miR-146a靶向SUMO1基因及miR-30a靶向NEDD4基因。后期将深入探讨特应性皮炎相关miRNA的调控机制。