EGF对紧密连接蛋白claudin的功能调控及对皮肤屏障的影响研究

We investigated the effect of long-term UV irradiation on skin barrier function, and the result revealed the increase of transepidermal water loss (TEWL), which was closely correlated with the down-regulation of claudin 1, indicating the key role of claudins in maintaining skin barrier function. Recent evidence indicated that claudins are regulated by a certain signaling trandcution pathways,including EGF signal pathway. It is well documented that skin barrier function may be injured by antitumor agents of epidermal growth factor receptor inhibitor (EGFRI), and claudins are speculated to play a role in the effect of EGF pathway on skin barrier function. However, the exact mechanism and molecular network of EGF pathway in controlling claudins are still unclear and need a further investigation. The study plans to regulate the expression of EGF in epidermal keratinocytes by incubation with exogenous EGF or EGFRI, and then evaluate the cellular barrier fuction using indexes such as transepithelial electrical resistance (TER) and paracellular permeability cell proliferation. The downstream ERK signal molecules are also to be investigated. Using TEWL, the essential factors as well as pathogenesis of EGF in regulating claudins and skin barrier function were further observed in vivo by animal experiment. Thus, the study will reveal the molecular mechanism of EGF on claudins and skin barrier function, and supply novel insights into clinical control of barrier-function associated cutaneous disorders.

课题组发现经皮水分丢失量（TEWL）在长期紫外线辐射之后增加，且与claudin1的下调相关，证实claudin蛋白在维持皮肤屏障功能中所起的作用。claudin蛋白的调控涉及包括EGF通路在内的一些信号转导通路，且肿瘤靶向药物EGFR抑制剂可引发皮肤屏障功能损伤，推测EGF通路可能通过对claudin蛋白的调控，实现对皮肤屏障功能的影响。但调控过程中的具体作用机制和分子网络目前不明，有待阐明。本研究拟通过外源性EGF和EGFR TKIs的添加影响表皮角质形成细胞中的EGF表达，以跨膜电阻和旁细胞渗透量为指标评估细胞屏障功能，并观察下游ERK途径中信号蛋白的表达改变，同时以TEWL为观察指标，在动物实验中验证EGF调控claudin蛋白和皮肤屏障功能的作用机制，以进一步阐明EGF调控claudin和皮肤屏障功能的分子机理，为屏障功能障碍性皮肤疾病的的临床诊治提供新的理论和研究依据。

表皮生长因子受体酪氨酸激酶抑制剂 (epidermal growth factor receptor tyrosine kinases inhibitors，EGFR-TKIs)作为一种分子靶向药物广泛地应用于肿瘤的靶向治疗。但在临床应用过程中，观察到其对皮肤的毒副作用，通常表现皮脂丰富部位丘疹脓疱性损害及皮肤干燥脱屑，提示可能存在皮肤屏障功能的损伤。为验证我们的设想，本课题组以表皮生长因子受体（EGFR）为靶点，通过添加外源性EGF和EGFR TKIs影响表皮角质形成细胞中的EGF表达，以跨膜电阻和旁细胞渗透量为指标评估细胞屏障功能，并观察下游ERK途径中信号蛋白的表达改变。然后以TEWL为观察指标，在动物实验中验证EGF调控claudin蛋白和皮肤屏障功能的作用机制，以进一步阐明EGF调控claudin和皮肤屏障功能的分子机理。体外细胞实验研究显示加入外源性的EGF作用后，HEK细胞EGF-ERK信号通路激活，ERK1，2磷酸化水平增高，claudin1，4蛋白表达增加并被活化，claudin2蛋白表达减少，磷酸化水平降低，细胞电阻增加，旁细胞渗透系数减小；外源性EGFR受体酪氨酸激酶抑制剂Gefitinib作用HEK细胞后，EGF-ERK信号通路被抑制，ERK1，2磷酸化水平降低，claudin1,4蛋白表达减少，磷酸化水平降低，claudin2蛋白表达增加，磷酸化水平升高，并且跨细胞电阻减少，旁细胞渗透系数增加。进一步通过动物实验证实了外源性EGF/EGFR-TKIs作用于小鼠后，小鼠表皮中EGF-RKF信号通路受到激活或抑制。claudin1，2，4表达的变化与细胞实验中观察到的结果一致，通过测量小鼠表皮含水量及经表皮水分丢失量证实小鼠表皮屏障功能受到EGF/EGFR-TKIs的影响。本研究初步阐明了EGF调控claudin和皮肤屏障功能的分子机理，为以屏障功能障碍为特征的皮肤病的临床诊治提供新的理论和研究依据，为减少靶向抗肿瘤药物对皮肤的副作用提供了新的防治策略，亦为今后研制更有效且皮肤毒副反应更小的抗肿瘤药物提供了有用的实验依据和理论基础。