EDA1/EDAR通过forkhead转录因子FoxA1调节3D重构汗腺泌汗功能的研究

EDA1/EDAR is one of the key signal pathways in the development of skin and its appendages. FoxA1 knockout mice and EDA1-mutant mice both reveals complete anhidrosis. Our previous studies showed that in the development of rat eccrine sweat glands, the expression of EDA1/EDAR appeared two peaks, namely sweat gland germ formation and secretory coil formation. FoxA1 was sharply up-regulated from secretory coil formation until adulthood. In 3D reconstituted eccrine sweat glands, the expression of FoxA1 was appeared at the time point that secretory coils began to differentiate, and FoxA1-positive cells co-expressed sweat secretion related functional protein, Na, K-2Cl co-transporter and Na, K-ATPase. Based on the above results, we propose the hypothesis that in the 3D reconstituted eccrine sweat glands, FoxA1 is the central for sweat secretion, which regulates sweat secretion through NKCC1 and Na，K-ATPase, and EDA1/EDAR may be involved in the activation of FoxA1. The study is based on the fact that we have confirmed that the 3D reconstituted eccrine swat glands had similar structures with the native ones and that small animal magnetic resonance imaging could be used to detect sweat secretion, and is involve methods such as magnetic resonance imaging, RNAi, qPCR, WB, three-dimensional cell culture and immunohistochemistry in order to study the sweat secretion function and the related mechanisms of 3D reconstituted eccrine sweat glands. It will lay a foundation for the repair and regeneration of eccrine sweat glands and also provides scientific basis for clinical treatment of burns.

EDA1/EDAR是调控皮肤附属器发育的关键通路，Foxa1敲除鼠和EDA1突变鼠均表现为无汗。我们前期研究显示：鼠汗腺发育中，EDA1/EDAR在胚芽期和分泌部形成时出现两次高峰；FoxA1在分泌部形成时迅速升高，直到成体。3D重构汗腺中，FoxA1在分泌部分化时开始表达，且FoxA1阳性细胞共表达泌汗相关功能蛋白NKCC1和Na，K-ATPase。因此我们提出“FoxA1是3D重构汗腺泌汗功能的关键作用点，FoxA1通过NKCC1和Na，K-ATPase等调节泌汗；EDA1/EDAR可能参与了FoxA1的激活” 的假说。本项目是在我们已经证实3D重构汗腺与在体汗腺具有相似结构和活体小动物磁共振可用于检测汗腺泌汗功能的基础上，采用磁共振、RNAi、qPCR、WB、三维培养、免疫组化等研究3D重构汗腺的泌汗功能及机制，其结果将为汗腺的修复和再生奠定研究基础，也为临床烧伤的治疗提供科学依据。

背景：外分泌汗腺在调节人体温度方面发挥着重要作用。严重大面积深度烧伤创面愈合后，由于大部分汗腺毁损，机体调节体温能力下降，患者常感闷热不适，严重影响了患者的身心健康与生活质量。EDA1/EDAR是调控皮肤附属器发育的关键通路。Foxa1敲除鼠，汗腺的形态发生正常，但汗腺却无功能，提示Foxa1是外泌汗腺汗液分泌的重要因子，如何调控Foxa1，再生有功能汗腺是烧伤科医生面临的挑战。.目的：探讨Foxa1在外泌汗腺发育及汗液分泌调控中的作用及机制。.方法：采用RNA-seq、组织学染色、免疫荧光组织化学染色、qRT-PCR和WB检测不同发育阶段大鼠外泌汗腺和人三维（3D）重构汗腺中EDA1/EDAR、Foxa1、Na+-K+-2Cl-协同转运蛋白1（NKCC1）和Na+-K+-ATPase（NKA）的表达，同时分析了这些基因的表达与汗腺形态发育和功能之间的趋势图。碘-淀粉发汗试验和小动物磁共振成像研究大鼠外分泌汗腺和人三维重构汗腺的泌液功能。通过生物信息学分析，以及Foxa1基因在工具细胞中的过表达和沉默，研究了Foxa1对汗腺汗液相关功能基因NCKK1和NKA的调控。.结果：在大鼠外分泌汗腺发育过程中，EDA1/EDAR出现两个高峰，即汗芽期和分泌部形成期；Foxa1 在分泌部形成时迅速增加，直到成年。在3D重构的外分泌汗腺中，Foxa1在分泌线部分化时表达，Foxa1阳性细胞共同表达汗液相关功能蛋白NKCC1 和NKA。NKCC1和NKA的mRNA丰度和蛋白质表达都显示出与Foxa1相似的累积趋势，表明它们存在潜在的相互作用。生物信息学分析表明，Foxa1可以通过其启动子区域中的保守基序与这两种蛋白质相互作用。Foxa1修饰细胞中Foxa1 的功能获得和功能丧失表明NKCC1和NKA的活性取决于Foxa1的存在。.结论：Foxa1是影响汗腺泌汗功能的关键因子。Foxa1通过NKCC1和NKA调节外泌汗腺的泌汗功能。EDA1/EDAR参与Foxa1的激活。.意义：为汗腺的修复和再生奠定研究基础，也为临床烧伤的治疗提供科学依据。