γ-分泌酶对角质形成细胞生长及EGFR信号通路调控机制的研究

Previous researches have found that gamma secretase was associated with Alzheimer's disease. In 2010, we firstly reported gamma secretase gene mutation was related with familial acne inverse (AI), but its mechanism was unknown, Currently, the pathogenic similarity and difference of these two diseases are focus of international academic community. Abnormal proliferation of keratinocytes was related to AI, and epidermal growth factor receptor (EGFR) not only regulated growth of epidermal cells but also was substrate of gamma secretase. So, in this study stable transfected HaCaT cell modles were established by short hair-pin RNA interfereing to silence different subunit of gamma secretase. Through fluorescent quantitative PCR, Western Blot, ELISA and laser confocal microscopy combined with whole genome microarray, changes of cell growth, adhesion and other biological functions as well as EGFR signal pathway in different gamma secretase subunits interferenced cells were compared. The above results were further verified in gamma secretase subunit gene knockout mice model and lesions of AI patients. This study may reveal the role of gamma secretase gene mutation in AI pathogenesis, and contribute to understanding the different molecular pathway of AI and Alzheimer's disease which both related to gamma secretase.

研究发现γ-分泌酶与阿尔茨海默病有关，2010年我们在国际上最早报道γ-分泌酶基因突变与家族性反常性痤疮相关，但具体机制不详，两种不同疾病发病机制的异同目前是国际学术界关注的热点。反常性痤疮的发生与角质形成细胞异常增殖相关，表皮生长因子受体（EGFR）及相关信号通路调控表皮细胞生长，同时EGFR还是γ-分泌酶的底物之一，故本课题拟通过shRNA分别干扰γ-分泌酶四个亚基建立稳定转染的HaCaT细胞模型，采用荧光定量PCR、Western Blot、ELISA、激光共聚焦显微镜等技术结合全基因组表达谱芯片，对比γ-分泌酶不同亚基干扰后细胞生长粘附等方面的变化以及与之相关的EGFR信号通路的改变，并选择患者皮损和1-2个γ-分泌酶亚基基因敲除小鼠模型进一步验证细胞实验结果。本研究可能揭示γ-分泌酶基因突变引起反常性痤疮的发病机制，有助于了解γ-分泌酶引起反常性痤疮与阿尔茨海默病分子通路的差异。

反常性痤疮，又称化脓性汗腺炎、毛囊闭锁三联症，是一种慢性复发性炎症性疾病，γ-分泌酶NCT亚基的突变证实与反常性痤疮密切相关。AI的病因尚不清楚，目前认为在遗传突变、免疫功能异常、性激素异常、吸烟、细菌感染等多因素共同作用下毛囊角化过度和毛囊上皮异常增生，导致毛囊角栓、膨胀、破裂并引发炎症反应，从而形成典型的临床表型。在NCT突变的家族性AI病人中我们发现NCT亚基的表达水平显著下调，以此为基础，我们构建了干扰NCT表达的角质形成细胞HaCaT细胞模型以及角质形成细胞特异性敲除γ-分泌酶亚基NCT的小鼠模型，用于探讨家族性反常性痤疮的发病机制。基于毛囊上皮细胞异常增生、角化过度导致的毛囊堵塞是反常性痤疮的始动因素，我们的研究结果发现NCT的表达缺失促进了角质形成细胞的异常增殖和分化，同时在细胞模型，小鼠模型以及病人临床样本证实参与角质形成细胞异常增殖和分化的PI3K-AKT信号通路被激活，EGFR通路被抑制，为家族性反常性痤疮的治疗提供可能的干预靶点和治疗思路。