基于层层自组装技术构建仿生人组织工程毛囊的研究

The dermal papilla cell (DPC) is the most important cell in construction of tissue engineering hair follicle(HF), due to its hair-inducing capacity. However, the inductive ability of DPCs tends to gradually lose during passaging once they are cultured in traditional 2D culture in vitro. In vivo, DPCs are physiologically embedded in extracellular matrix (ECM), forming into a multi-cellular spheroid tissue that exhibiting a unique 3D organization. Since Layer-by-layer self-assembly (LBL) is an ultrathin ECM nano-fabrication technique to create a 3D biomimetic microenvironment. Recently, we have developed a single mouse DPC-based LBL encapsulation model (LBL-DPC) by utilizing the LBL technique, and DPC hair-inductivity was enhanced when defined growth factor was loaded into the LBL nano-structure. In our latest work, we have preliminarily established a biomimetic model of mouse LBL-DPCs multicellular spherical microtissue based on chemical crosslinking. Thus, we want to construct nanometer ultra-thin ECM for human DPC for LBL-DPCs microtissue, which co-culture with human HF stem cell to reconstruct bionic microenvironmentand tissue engineering HF. It will not only provides a valuable platform for exploring the biology and mechanism of human HF, but also confers a potential therapeutic strategy for treating hair loss diseases.

人毛乳头细胞（DPC）具有毛囊诱导功能，是构建人组织工程毛囊重要的种子细胞。但是，DPC经体外常规二维培养后会逐渐丧失诱导能力。正常人体内的DPC聚集呈球状，周围被丰富的细胞外基质包裹（ECM）。层层自组装(LBL)技术被广泛用于构建细胞纳米仿生ECM及微环境。我们在近期研究中以LBL技术成功构建出了鼠单个DPC的仿生纳米超薄ECM包裹模型(LBL-DPCs)，为单个DPC提供了合适的微环境。此外，我们在包裹层中载入特定生长因子后能一定程度上增强DPC的毛囊诱导能力。在后续实验中，我们基于化学交联技术构建出了仿生化的鼠LBL-DPCs多细胞球状微组织，这一定程度上模拟了DPC体内微环境。基于此，我们拟采用 LBL技术构建人DPC纳米超薄ECM，并以化学交联技术构建出仿生人DPC微组织，此后与人毛囊干细胞三维共培养，以期重建出人组织工程毛囊。

脱发是一种非常常见的毛发相关性疾病，目前临床上不管是手术还是药物，治疗效果均有限。组织工程毛囊再生是治疗脱发最有前景的治疗手段。虽然目前尚未在体外构建出完整的组织工程人毛囊，但研究表明三维培养有助于细胞形成类器官样结构。组织学显示人体内的毛乳头细胞（DPCs）处于丰富细胞外基质(ECM)的包裹之中。我们在前期研究中以层层自组装(LBL)技术成功构建出了鼠单个DPC的仿生纳米超薄ECM包裹模型(LBL-DPCs)，为单个DPC提供了合适的微环境。此外，我们在包裹层中载入特定生长因子后能一定程度上增强DPCs的毛囊诱导能力。本项目我们拟采用 LBL技术成功构建人DPC纳米超薄ECM，并以化学交联技术构建出仿生人DPCs微组织，此后与毛囊干细胞三维共培养，以重建出人组织工程毛囊。经过一些列的研究，首先，我们成功实现了人毛囊干细胞（HFSCs）和人毛乳头细胞（DPCs）的分离、培养；接着通过LBL技术体外构建人DPC单细胞仿生纳米 ECM 包裹模型（LBL-DPC），并应用化学交联构建 LBL-DPCs 微组织；同时我们还建立了体外人HFSCs的培养模型，应用LBL进行包裹为其构建细胞外基质，载入信号分子(BMP)及细胞因子（FGF/VEGF）等，并探讨了TGF-β2调控HFSCs分化的作用机制；最后将HFSCs和DPCs以一定比例在体外和体内实现毛囊重建及再生。以上研究为毛囊再生及组织工程毛囊构建的进一步研究提供了良好的理论和研究基础。