EGFR在人外泌汗腺三维模型构建中的作用及机制

For patients with extensive deep burns, the reconstruction of eccrine sweat gland to restore the sudomotor function is very important. Three-dimensional culture models are physiological correlated culture modes, which recapitulate the dynamic and interactive signaling in tissue morphogenesis. The in vitro monolayer culture technique, skin specimen detection and animal experiment showed that EGFR played key roles in the development, function maintain and the tumorigenesis of sweat gland. We have successfully constructed the in vivo and in vitro 3D culture model of sweat glands by culturing sweat gland cell in extracellular matrix. Our preliminary studies show that the EGFR inhibitor inhibits the formation of the 3D structure. On these above grounds, we raise the hypothesis that EGFR is the central element in the ECM-induced construction of three-dimensional sweat gland models. The extracellular signals activate EGFR and then EGFR activate the downstream signaling pathway, which result in gene transcription, cell proliferation, differentiation, apoptosis and ploarity and then the construction of the 3D structure of sweat glands. The research is to study on the roles and mechanisms of EGFR in the construction of three-dimensional culture models of human eccrine sweat gland by immunohistochemistry, RT-PCR, western, co-immunoprecipitation, gene transfection, RNAi, RNAi rescue experiment, and so on. The results will provide new strategies and models as well as the baseline data on future developments of eccrine sweat gland repair and regeneration.

汗腺的重建对于恢复大面积烧伤引起的泌汗功能降低有重要意义。三维培养模式（3D）是与生理相关的培养模式，它捕获了建立组织形态发生的动态和交互的信号。体外单层培养技术、皮肤标本检测和动物实验研究发现EGFR在汗腺发育、功能维持和汗腺肿瘤的发生中起着重要作用。我们利用细胞外基质成功地构建了汗腺细胞体内外3D培养模型，同时在预实验中发现：用EGFR抑制剂后，汗腺细胞不能重构3D结构。基于上述结果我们提出"EGFR是细胞外基质诱导汗腺细胞重构汗腺三维结构的核心成份。EGFR活化后，又激活下游的信号通路，引起基因转录，细胞增殖、分化、凋亡和极性，从而重构汗腺三维结构"的假说，本项目拟应用免疫组织化学、RT-PCR、western、免疫共沉淀、基因转染、RNAi、RNAi补救实验等手段和方法研究EGFR在汗腺3D模型构建中的作用及机制，其结果将为汗腺的修复和再生提供未来发展的新模型、新策略和基准数据。

外泌汗腺的重建对于恢复大面积烧伤引起的泌汗功能障碍具有重要意义。三维培养模式（3D）捕获了组织形态结构发生的动态和交互信号。我们利用细胞外基质对细胞的诱导分化作用，重构了汗腺的3D结构，并对EGFR在汗腺三维重构中的作用和机制进行了研究。结果显示：3D重构的汗腺从形态(类球状或类管状结构，具有腔和基底膜)、细胞分化（分化成类分泌部和导管部；5种细胞类型）、标志抗原表达（K5/7/8/14/15、α-SMA、CEA）、细胞连接蛋白（ZO-1/2、claudin-1、桥粒蛋白1/2和上皮钙连接蛋白）、细胞极性蛋白（β-cateinin、collagen Ⅳ、 ZO-1、F-actin）功能蛋白表达（Na-K-ATPase β、Na-K-2Cl-共转运体1、水通道蛋白5）、血管营养（CD31、α-SMA）、神经支配（蛋白基因产物9.5， 酪氨酸羟化酶，血管活性肠肽）、泌汗功能上，均类似于在体汗腺。EGFR在汗腺的3D重构中起着重要作用：汗腺3D结构形成早期，抑制EGFR（1μM AG1478）或β1-integrin1(10μg/ml β1-integrin功能阻断抗体)或RAS/ERK(1μM U0126)通路，汗腺细胞均不能重构3D汗腺；去除EGF后，不影响汗腺3D结构的形成；汗腺细胞聚集后，AG1478对汗腺3D结构的形成无明显影响。EGFR通过β1-integrin途径，而非传统的受体-配体途径激活，随后通过下游的RAS/ERK通路，引起细胞的粘附和聚集、增殖、自噬、分化；EGFR对凋亡无影响。本研究为汗腺的发育、细胞分化和调控、以及再生提供了新模型、新思路和新策略。