外胚层附属器是来源于胚胎原始皮肤的功能性附件，研究外胚层附属器再生的问题可以解决烧（创）伤后移植皮肤不能出汗和没有毛发生长。Wnt/β-catenin 和EDA/EDAR通路在外胚层附属器发生中起重要的作用。间充质干细胞（hMSCs）目前被用来诱导再生外胚层附属器。但是，存在的一个难题是hMSCs难于跨外胚层分化。控制细胞增值的Hippo通路多方式地与Wnt/β-catenin通路相互作用，并且信号汇聚在下游效应子YAP。在细胞核内，YAP可能通过结合β-catenin促进hMSC向外胚层附属器分化。SIRT1介导YAP和β-catenin都可以去乙酰化。科学问题是： SIRT1去乙酰化修饰对YAP和β-catenin相互作用的影响，及其对增强外胚层附属器基因表达的机制和结果尚不清楚。拟采用蛋白质组学等多种方法检测SIRT1介导YAP/β-catenin的相互作用、转录活性以及分化结果。

Ectodermal appendages is a functional accessories from embryonic skin including hair follicles, sweat glands, and etc. The issue on the regeneration of ectodermal appendages can be used to solved the problem for skin graft without sweating and hair growth after burn (create) injury. Wnt/β-catenin and EDA/EDAR pathway both play important role in the occurrence of ectodermal appendages. Mesenchymal stem cells (hMSCs) are used to induce regeneration of ectodermal appendages in vitro. However,an problem is difficult for hMSCs to across the ectodermal to differentiation. Another signal pathway is Hippo/YAP,controlling cell proliferation,interacts with Wnt/β-catenin pathway in multiple ways, and the signal is gathered in YAP, the downstream effector of Hippo pathway. In the nucleus, YAP may be combined with β-catenin to enhace hMSC differentiated into ectodermal appendages. As it has been reported that SIRT1 mediated YAP and β-catenin to deacetylation. The scientific question is: The mechanism and the results of deacetylation modification by SIRT1 on YAP and β -catenin interactions, and on the enhancement of ectodermal appendages gene expression is unclear. As a result, we will investigate YAP/beta-catenin interactions, transcriptional activity of YAP/beta-catenin ,and differentiation results by SIRT1 mediated.

按照研究进度计划，围绕“SIRT1去乙酰化修饰对YAP和β-catenin相互作用的影响，及其对增强外胚层附属器基因表达的机制”的核心假设，我们完成以下的工作：（1）胶原酶消化法分离培养原代汗腺细胞，以及淋巴分离液分离原代培养hMSCs，建立hMSCs跨胚层转分化汗腺细胞模型；（2）应用荧光素酶报告系统发现, 在敲除SIRT1的hMSCs（SIRT1-/-）细胞, YAP转录活性减少, β-catenin转录活性减少。激光共聚焦观察YAP/β-catenin相互作用；（3）在EDA诱导hMSCs定向外胚层附属器分化的体外细胞模型中，SIRT1对细胞核内YAP和β-catenin转录活性的效应的研究，经 realtiemPCR检测发现实验组比较突变的对照组外胚层附属器发生相关基因CEA, CK8，CK18，CK19的表达升高。研究结论:SIRT1作为一个变阻器（rheostat）通过去乙酰化修饰调节YAP和β-catenin控制hMSCs向外胚层附属器汗腺分化。（4）研究成果有两篇相关的SCI 论著在修回中，待发表后及时向基金委网站上传。