丝聚蛋白、屏障功能及金黄色葡萄球菌的协同关系在AD发病中作用

TThe pathogenesis of Atopic dermatitis (AD) remaimed unsurtain. Many recent studies have revealed the key roles played by filaggrin deficiency, barrier abnormality and high amount of S.aureus colonized on the skin in the pathogenesis of atopic dermatitis. Neither of the three key causes alone suffices to elicit AD, where three factors intertwine to provoke AD. We want to further investigate the synergistic effect between barrier abnormality,filaggrin deficiency and S.aureus colonization, and how they impact the skin immunologic reaction and derect it toward a Th2 immunophenotype inflammation..our previous researchs by using non-invasive skin assessment,we found that AD patients had poor barrier function,and topical used of moisturizer could alleviate the inflammation. The research on FLG mutation flaky tail mices shown that their skin exhibiting increased bidirectional, paracellular permeability of water-soluble xenobiotes due to impaired barrier function. This barrier abnormality correlates with reduced inflammatory thresholds to both topical irritants and haptens. When exposed to low dose of irritants, the flaky tail mice developed a severe AD-like, th2 immunophenotype dermatosis. All these works support that filaggrin deficiency and abnormal barrier function can impact the inflammation reaction in the skin...In this study, we will employed two pre-existing models, the oxazolone-induced AD murine model and artificial destroyed skin barrier murine model,which exhibit barrier abnormalities.By utilizing the two models, we want to observe the correlation between barrier integrity and the colonization of S.aureus, and furthe investigate the inflammtion pattern in the mices skin toward S.aureus.Then we will aritificially restore the barrier function by using moisturizer or occlusion and observe the changes in the amount of S.aureus and skin inflammation, which can further verify our propose that the barrier abnormal facilitate the S.aureus to colonize and cause Th2-dominant inflammation in the pathogenesis of AD...We will further use RNA interference to achieve filaggrin knockdown in HaCaT cells and stimulated it with membrane components of S.aureus, then subsequently compared the expression of pattern-recognition receptors, specifically Toll-like receptors (TLRs) and proinflammatory cytokines.These will help to elucidate the possible impact of filaggrin deficiency on skin immunological reaction towards the S.aureus. And how and why AD patients can develop Th2-dominant responses in the presence of huge amount of S.aureus..The results of above study is useful for elucidating the interaction between filaggrin deficiency, barrier abnoraml and S.aureus in pathogenesis of atopic dermatitis.They will also enlight the prevention measures and treatment for other pathological microorganisms on the skin and mucosa.

目前AD发病机理不清，丝聚蛋白、屏障功能及金黄色葡萄球菌是三大致病因素。但这三种因素单独作用都不能导致AD。本课题拟研究三者的协同作用：重点是丝聚蛋白和屏障功能缺陷如何影响皮肤免疫活性，针对金黄色葡萄球菌产生Th2为主炎症反应及可能的作用通路。我们前期通过无创性皮肤测试发现AD患者屏障功能受损，保湿剂可以减轻炎症反应。丝聚蛋白基因突变的层状尾小鼠屏障功能受损，皮肤对大分子物质通透性增加，对刺激物免疫反应增强，产生Th2炎症反应。这些提示丝聚蛋白和屏障功能对皮肤的炎症反应有影响。我们将在AD和人为破坏屏障的小鼠模型上，观察皮肤对金黄色葡萄球菌炎症反应特点;观察丝聚蛋白siRNA敲低的HaCaT细胞对金黄色葡萄球菌膜成分产生的细胞因子等反应特点，探讨丝聚蛋白和屏障功能与金黄色葡萄球菌之间的协同作用及可能的通路。本课题不仅有助于阐明AD的发病机理，并为防治皮肤黏膜致病微生物感染提供了新的思路。

遗传过敏性皮炎（atopic dermatitis, AD）是一种常见的皮肤炎症性疾病。我国AD患病率为3.07%，北京达4.75%。.AD的发病机理还不清楚。研究都指向三个重要因素：丝聚蛋白减少、屏障功能损伤及金葡菌定植。但是这三个因素单独存在时都不一定会引发AD。假说：在AD中这三个致病因素之间存在协同作用，丝聚蛋白缺陷导致皮肤屏障功能损伤会促进金葡菌定植；丝聚蛋白缺陷和相应的皮肤屏障功能损伤可以直接影响皮肤的免疫功能，导致在金葡菌的刺激下出现Th2为主导的炎症反应。.我们想通过下列实验证实假说：1.在OX反复刺激诱导的AD小鼠模型上，外用保湿剂、封包等帮助皮肤屏障功能恢复，观察屏障功能完整性与免疫之间的关系。2.在上述AD小鼠模型的皮肤表面接种金葡菌，观察菌量和皮肤炎症反应的类型和程度。3. 通过丝聚蛋白siRNA敲低的培养原代角质形成细胞，观察它在遇到金葡菌菌膜成分的刺激后，在炎症因子分泌方面的变化。反复涂抹OX建立的AD小鼠模型有典型的湿疹表现；TEWL升高，皮肤表面pH值升高和角质层含水量降低；组织病理示表皮增生，海绵水肿，真皮多数淋巴细胞浸润，免疫组化显示其是Th2淋巴细胞，真皮中还有大量的肥大细胞。AD小鼠皮肤中IL-4、IL-13表达量较正常小鼠明显上升；TSLP、 TLR2、S100A8/S100A9的表达也上调，表皮抗菌肽BD3和cathelicidin的表达下调。外用保湿霜后，AD小鼠背部红斑、水肿明显好转，鳞屑减少。TEWL和pH值降低，角质层含水量增高，pH值恢复至正常小鼠水平。对应的IL-4、IL-13 和TLR2的表达量均明显下降。在AD小鼠模型和正常小鼠上接种金葡菌，AD小鼠皮肤表面金葡菌菌落数量明显高于正常小鼠。保湿和封包等恢复皮肤屏障功能的手段可以显著减少金葡菌定植。 丝聚蛋白siRNA转染的原代角质形成细胞filaggrin基因的表达量明显下降。IL-4，IL-1a，TNF-a表达量较对照组明显上升，MyD88及TLR-2表达量较对照组也有明显上升。本研究证明：皮肤屏障功能的完整性会直接影响皮肤的免疫状态，包括皮肤的天然免疫活性如表皮抗菌肽的表达水平，以及皮肤在面对致病微生物时的免疫反应类型。通过影响丝聚蛋白的表达情况也可以直接影响皮肤的免疫状态，包括细胞因子和TLRs表达及其对于金葡菌表面膜成分的免疫反应。