人羊水干细胞向功能性汗腺细胞分化及其调控机制的研究

The differentiation from stem cells into sweat gland cells (SG) is still a problem to be solved, and its related mechanism is not clear. Under certain inducing conditions, several hAFS cell lines set up in our laboratory can differentiate into the cells expressing epithelial cell-specific gene of K14, K18, K19, SG cell marker gene Eda / Edar, and with the ability of forming sweat gland-like structure both in 3D gas-liquid culture system and the animal model. On the basis of preliminary work, this project aims to do further explore on differentiation from hAFS to SG cells and its related regulation mechanism. (1)To identify hAFS-SG cells from different aspects as observing and detecting function associated marker proteins, ultrastructures, and the sweat gland-like structures both in 3D gas-liquid system and animal model and the perspiration experiments in the animal model also to provide more valuable experimental datas for the clinical use of the hAFS-SG cells in sweat glands reconstruction of the skin. (2)To do further explore on the role and regulation mechanism of different signaling pathways on the differentiation of hAFS-SG through regulating the expression of SG cell marker gene Eda / Edar, which will lay the foundation for elucidating the molecular mechanisms of cell differentiation hAFS-SG, take it together, this project has great value both in theoretical study and clinical application.

干细胞分化为汗腺细胞（sweat gland cells, SG）至今仍是待解决的难题，且其分化机制尚不明确。本实验室建立的多株人羊水干细胞（human Amniotic Fluid Stem Cells, hAFS）细胞株，在特定诱导条件下，可分化为表达上皮细胞特异性基因K14、K18、K19和SG细胞标志基因Eda/Edar、在3D气液培养体系及裸鼠体内形成汗腺样结构的细胞，在该工作基础上，本项目旨在：(1)从与功能直接相关的标志蛋白及超微结构入手，结合体外3D气液培养和体内排汗实验，从不同层面对hAFS-SG细胞进行鉴定，为该细胞在临床皮肤汗腺重建中的运用提供有价值的实验数据。(2)以SG细胞特异性标志分子Eda/ Edar为切入点，深入探讨不同信号通路在hAFS-SG细胞诱导分化过程中对其表达的作用及调控，为阐明hAFS-SG细胞分化的分子机制奠定基础，具有理论原创和潜在运用价值。

hAFS来源广泛、取材方便，具有极强的分化潜能但不致畸，同时它免疫原性低，且表达一系列上皮来源细胞的特异性基因，在汗腺细胞定向诱导分化过程中，是具有潜在临床运用价值的、理想的种子细胞，本项目首先建立了人汗腺细胞的体外分离、培养、纯化体系，并进行有效扩增，完成对汗腺细胞的鉴定，为进一步研究汗腺细胞提供了理论依据，其次对现有的汗腺细胞分化方案进一步完善，对hAFS-SG细胞标的汗腺细胞特异性标志分子EDA,EDAR,CEA进行检测，完成hAFS-SG细胞生物学特性鉴定，同时采用3D培养体系中形成管状结构、及管状结构对汗腺细胞特异性标志分子EDA, EDAR, CEA, K8, K18，Na-K ATPase，EMA等表达的检测、hAFS-SG细胞对Ach的受体的表达以及在Ach作用下细胞内游离Ca2+浓度的检测，以及将hAFS-SG细胞注射入裸鼠肩部脂肪垫，电镜下观察可见该处可以形成汗腺腺泡样结构，从而完成hAFS-SG细胞分泌功能进行鉴定，最后对hAFS-SG细胞分化过程中Wnt、BMP及ERK信号通路的作用进行分析，实验结果表明EDA/EDAR信号通路的下游通路shh信号通路在hAFS-SG细胞分化过程中发挥一定的作用，此外,我们成功构建了汗腺细胞来源的iPS细胞，并以SG-iPS细胞为研究对象，对汗腺细胞的分化机制进行进一步的研究，实验结果表明条件培养基中BMP4和HGF分别由SG和SG-Fib分泌，是SG-iPS分化为汗腺样细胞的过程中起关键作用的分子。综上，本项目所获研究结果将为hAFS-SG细胞在临床皮肤汗腺重建的运用，提供更多有价值的理论依据和实验基础。