利用跨谱系转化关键因子诱导间充质干细胞分化为汗腺细胞的研究

Sweat gland regeneration plays the important role in the process of wound repair and remodeling. Stem cells provide a novel approach to resolve this conundrum. Our previous research has proved the possibility that mesenchymal stem cells could acquire phenotype of sweat gland cells after in vitro induction. However, the molecular mechanism of this differentiation remains unclear, limiting the effort to promote the poor differentiation efficiency. Current project is aimed to screen and sort out the key transcription factors for specification of the sweat gland cells by ChIP-Seq technology. Through regulation of these transcription factors and critical signaling molecules involved in mesenchymal-epithelial transition (MET), we will try to enhance the specific reprogramming efficiency of mesenchymal stem cells into sweat gland cells, and to establish the stable production scheme of induced sweat gland cells. In further study, we will evaluate the effects of induced sweat gland cells derived from mesenchymal stem cells on sweat gland regeneration via in vitro 3-D model and in vivo animal model. This will be the first study which stems from the perspective of sweat gland development and mechanism for transition between different germ layers to explore and to establish the system of sweat gland regeneration based on cell reprogramming and 3-D microenvironment, and to provide the foundation of establishing clinical projects of post-traumatic sweat gland function recovery. This study will also offer valuable information for the regeneration of other complex tissues and organs, by application of combined mode-based strategy of intracellular reprogramming events and extracellular microenvironment education.

汗腺再生在皮肤创伤修复和重建过程中的作用至关重要，干细胞为解决汗腺再生的难题提供了新的途径。申请人研究组已证明骨髓来源间充质干细胞（MSC）经体外诱导可分化为具有汗腺细胞表型的细胞。然而，MSC转变为汗腺样细胞的分子机制尚不清楚，从而限制了转化效率的提高。本项目拟采取ChIP-Seq方法筛选汗腺细胞特化的关键转录因子，通过调节这些转录因子和间充质-上皮转化（MET）的信号分子，达到利用其协同作用提高MSC向汗腺细胞特异性重编程的效率，建立稳定的诱导性汗腺细胞生成方案; 进一步通过成熟的体外三维和体内动物模型，评价MSC源性的诱导性汗腺细胞在汗腺再生中的作用。本项目首次从汗腺发育和跨胚层转化机制角度，探索和建立基于细胞重编程和三维微环境的汗腺再生体系，为建立烧创伤后汗腺功能恢复的临床治疗方案提供基础，也为其他复杂组织器官缺损的再生研究提供基于细胞内重编程和细胞外微环境组合模式的策略参考。

汗腺再生是皮肤及其附属器损伤后修复与再生过程中的关键环节。转录因子重编程技术对再生医学发展的推动具有光明前景，然而效率低及机制不明确等因素成为限制该技术应用的重要因素。在本项目的支持下，团队顺利完成了以跨谱系转化关键因子诱导成体细胞分化为汗腺细胞以及体外3D微环境精准调控MSC定向分化为汗腺细胞等一系列再生创新策略研究。该研究完成过程中的重要发现包括：①高通量筛查并寻获可诱导特化汗腺细胞的核心转录因子：根据表皮细胞和汗腺细胞HiSeq2500 RNA测序，选取汗腺细胞中表达量为表皮细胞中表达量3倍以上（包含3倍）的转录因子。通过GO分析和RT-PCR验证，选定3个转录因子FoxC1, Irf6, Tfcp2l1；其中FoxC1转染表皮细胞可以显著诱导汗腺标志物mRNA、蛋白表达并引起细胞增殖能力的变化；体外及体内结果均证实FoxC1诱导汗腺细胞具有发汗功能。②FoxC1过表达致汗腺分化的分子机制： FoxC1通过激活Wnt，进而启动Wnt-Eda-Shh/NF-kB这一汗腺发育的级联通路，诱导汗腺相关基因表达。③利用生物3D打印技术建立可诱导MSC定向分化为汗腺的微环境：光镜下可观察到汗腺发生过程（从细胞凝聚团到汗腺样结构）；免疫荧光检测汗腺特异标志物检测（K8+/K18+）、汗腺钠钾ATPase及钙离子摄取阳性，且分化过程中有MET发生。④微环境诱导机制探讨：对可诱导MSC的微环境蛋白进行差异比较，通过GO功能分析筛选出Cthrc1为目标蛋白。Cthrc1抗体注射小鼠出生后汗腺发育减缓及发汗功能减弱进一步确证其特异性。以上研究从汗腺特化和定向分化阶段的特征入手，在汗腺谱系细胞中归集特异性强的转录因子以及微环境诱导特异蛋白，达到了高效利用成体干细胞重编程诱导汗腺细胞的目的，并深入探讨了相关机制，为进一步面向临床治疗的应用研究奠定基础。