从表观遗传角度探讨特应性皮炎角质形成细胞TSLP基因异常表达的机制

Atopic dermatitis (AD) is an inflammatory, chronically relapsing, non-contagious and pruritic skin disorder, the pathogenesis and pathophysiology of AD is complex and not yet understood. Previous studies have showed that the Th1/Th2 imbalance plays an important role in AD pathogenesis, and recent research show that this imbalance is related to the highly expressed thymic stromal lymphopoietin (TSLP) in keratinocytes. A variety of environmental factors can lead to the high express of TSLP, our previous study show that DNA hypomethylation of TSLP promoter and overexpression of TSLP in keratinocytes treated with 5-aza.It is not clear whether the environmental factors involved in the pathogenesis of AD by overexpress the TSLP through hypomethylation of TSLP regulatory sequences. This project focus on the regulation of TSLP by DNA methylation, study on the differences of DNA methylation status in TSLP gene promoter of keratinocytes between AD and normal control, the effect of abnormal DNA methylation to the expression of TSLP, the relationship between environmental factors induced TSLP expression and the DNA methylation modification. We investigate the effect of DNA methylation to the TSLP expression of keratinocytes from the perspective of epigenetic for the first time. This helps to reveal the epigenetic mechanism of AD pathogenesis, and provide new ideas for the protection of AD.

特应性皮炎（AD）是常见的慢性变态反应性皮肤病，发病机制尚不十分清楚，已有研究表明Th1/Th2失衡在AD发病中起重要作用，而最近研究表明AD患者Th1/Th2失衡与皮肤角质形成细胞高表达胸腺基质淋巴细胞生成素（TSLP）密切相关。多种环境因素可致TSLP表达升高，我们的前期研究发现DNA甲基化调控TSLP基因转录表达，DNA甲基化抑制剂使人角质形成细胞TSLP表达增加。是否环境因素引起TSLP基因启动子调控序列低甲基化，使TSLP基因表达增加导致AD发病呢？本课题以DNA甲基化对TSLP的调控为着眼点，研究AD角质形成细胞TSLP基因启动子DNA甲基化状态；DNA甲基化异常对TSLP表达的影响；环境因素诱导TSLP表达增加与DNA甲基化异常修饰的关系。首次从表观遗传学角度研究DNA甲基化对人角质形成细胞TSLP蛋白基因表达的调控，有助于揭示AD发病的表观遗学机制，为防治AD提供新的思路。

特应性皮炎（AD）是常见的慢性变态反应性皮肤病，发病机制尚不十分清楚，已有研究表明Th1/Th2失衡在AD发病中起重要作用，而最近研究表明AD患者Th1/Th2失衡与皮肤角质形成细胞高表达胸腺基质淋巴细胞生成素（TSLP）密切相关。本课题以DNA甲基化对TSLP的调控为着眼点，研究AD角质形成细胞TSLP基因启动子DNA甲基化状态；DNA甲基化异常对TSLP表达的影响。在本研究中，我们取正常人与AD患者皮损组织，Real-time-PCR检测角质形成细胞中TSLP mRNA表达水平；免疫组化检测角质形成细胞中TSLP 蛋白表达水平；亚硫酸氢盐测序检测TSLP基因启动子调控序列甲基化状态。发现AD患者皮损组织角质形成细胞TSLP mRNA及蛋白表达水平显著升高，同时其基因启动子调控序列DNA甲基化水平显著降低。以5-azaC处理体外培养的人角质形成细胞，使其DNA低甲基化，与对照组相比，处理组的角质形成细胞TSLP mRNA及蛋白表达水平明显升高。以双链RNA处理培养的人角质形成细胞，发现处理组TSLP mRNA及蛋白表达水平明显升高,但DNA甲基化水平无明显差异。以甲基化化酶处理TSLP基因启动子区包含CpG岛的DNA序列，使其高甲基化，转染至人角质形成细胞，同时共转染海肾荧光素酶报告载体pRL - CMV，荧光闪烁计数器检测荧光强度，发现高甲基化组荧光强度明显低于对照组。本研究证实AD患者皮损角质形成细胞中TSLP基因启动子调控序列低甲基化导致该基因过度表达，而高甲基化可以抑制TSLP的表达，首次从表观遗传学角度研究DNA甲基化对人角质形成细胞TSLP蛋白基因表达的调控，有助于揭示AD发病的表观遗学机制，为防治AD提供新的思路。