从表观遗传修饰Wnt通路研究凉血潜阳法对银屑病“血热证”调控作用的分子生物学机制

Based on the clinical experience of the XiaShi surgery treatment of skin diseases and the previous studies, it's confirmed that cooling blood and restrain Yang treatment could inhibit keratinocyte dysplasia. According to the epigenetic research progress, we put forward the hypothesis on the relationship between the mechanism of cooling blood and restrain Yang treatment in blood heat syndrome of psoriasis and the role of DNA methylation of Wif-1 in the Wnt pathway. Using methylation-specific PCR, RT-PCR, immunohistochemistry, Western blot, Wif-1 methylation status and gene expression of human keratinocytes as well as silence effect of DNA methylation in Wif-1 expression will be detected to reveal the molecular mechanisms of DNA methylation of the Gene Wif-1 in the Wnt pathway. Confirmed and evidence to the contrary of blood heat syndrome through recipe measurements Syndrome type. The project also aims to clarify cooling blood and restrain Yang treatment on the suppression of excessive proliferation and improvement of the lesions situation related to the adjustment of Wif-1 gene expression and the level of CpG island methylation. In vitro experiments, using the method of RNA interference and demethylation method, it will explain the role of Wif-1 expression silence effect on biological behavior of the Wnt signaling pathway such as the regulation of cell proliferation, cell cycle and apoptosis. The project may confirm that there may be epigenetic modificative effects in the Wnt signaling pathway related to the development of blood-heat syndrome of psoriasis vulgaris , and investigate the cell molecular biology mechanism of cooling blood and restrain Yang treatment and its inherent laws.

根据夏氏外科治疗皮肤病临床经验，在研究明确凉血潜阳法抑制角质形成细胞异常增生基础上，结合表观遗传进展，提出"凉血潜阳法作用银屑病血热证机制的关键环节与DNA甲基化修饰Wif-1调控Wnt通路密切相关"假说。项目运用MSP，RT-PCR，免疫组化，Western blot技术检测人角质形成细胞Wif-1甲基化状态和基因表达，评价DNA甲基化修饰对Wif-1表达的沉默效应，揭示Wif-1基因启动子甲基化致Wnt通路异常活化分子机制；同时阐明清热凉血法抑制过度增生、改善皮损与调节患者Wif-1基因表达和CpG岛甲基化程度关系，并运用以方测证印证、反证。离体实验采用RNA干扰和去甲基化方法，通过功能学研究评价Wif-1表达沉默对Wnt信号通路调控细胞增殖、周期及凋亡等生物学行为影响。明确表观遗传修饰Wnt通路参与银屑病"血热证"发生发展过程，探讨凉血潜阳法作用的细胞分子生物学机制及其内在规律。

银屑病是具有遗传素质个体在内外环境因素作用下，由T细胞介导的自身免疫性皮肤病。研究证明表观遗传在银屑病发生、发展中发挥了重要作用。本研究在项目组前期研究证实凉血潜阳抑制角质形成细胞异常增生基础上，结合银屑病表观遗传研究进展，验证“凉血潜阳法作用寻常型银屑病血热证机制的关键环节与DNA甲基化修饰Wif-1调控Wnt通路密切相关”科学假说。临床研究纳入受试者80例，凉血潜阳法辨证加减治疗组总有效率（87.50%）高于固定方治疗组（84.21%），凉血潜阳法疗效肯定。基础研究采用在体和离体实验相结合。在体实验证实银屑病患者角质形成细胞Wif-1基因启动子活性受DNA甲基化调控（CpG位点为366和240）；与正常受试者相比，银屑病血热证组、非血热证组甲基化水平明显升高，有统计学差异（P＜0.05）；与正常受试者比较Wif-1 mRNA和蛋白表达表达均下降；运用凉血潜阳法治疗以方测证，疗效愈显甲基化水平降低，无效甲基化水平上升，印证与反证凉血潜阳法抑制过度增生、改善皮损与调节患者Wif-1基因CpG 岛甲基化程度和基因表达关系。离体实验以正常受试者和寻常型银屑病血热证患者血清干预的HacaT细胞为研究对象，采用Wif-1 shRNA转染和去甲基化方法，明确高甲基化致Wif-1表达沉默对Wnt信号通路调控细胞增殖、凋亡等生物学行为影响，从而揭示银屑病血热证患者Wif-1第240位碱基和第366位碱基CpG高甲基化状态致Wnt通路异常活化的分子机制。研究有利于拓展银屑病药物治疗新领域，对临床寻常型银屑病血热证的辨证施治具有积极意义。