基于仿生细胞微环境构建人组织工程毛囊的研究

For severe cases of hair loss or alopecia, regeneration of hair follicles (HFs) by bioengineering is a promising future treatment alternative. To induce neogenesis of HFs, rodent DPCs has been demonstrated to be able to transdifferentiate epidermal keratinocytes into HF structures when DPCs are delivered precisely to the dermal-epidermal junction. However, owing to specific differences and ethical issues on human trials, we have yet realized the regeneration of human HFs. Physiologically, DPCs are embedded in extracellular matrix (ECM) rich in basement membrane proteins, including ?bronectin, collagen IV and laminin. Cells interact with neighboring cells and the ECM to establish a unique three-dimensional organization in vivo. These cell-cell and cell-ECM interactions form a complex communication network of biochemical and mechanical signals which are critical for normal cell physiology. When cells are cultured on two-dimensional plastic substrata, important signals controlling programs of cell proliferation, metabolism, differentiation and death responsible for the formation for correct tissue-specific architecture and function are lost. Thus, designing three-dimensional, physiologically relevant culture models, we can recapitulate some crucial aspects of the dynamic and reciprocal signaling necessary for establishing and maintaining tissue specific morphogenic programs. A simple in vitro system for the ECM is the Matrigel basement membrane matrix, extracted from the EHS mouse sarcoma, which consists primarily of laminin, collagen IV, heparin sulfate proteoglycans, and growth factors. Matrigel has been successfully applied in three-dimensional cell cultures for the purpose of elucidating the establishment of mammary cell morphogenesis and angiogenesis. Our previous studies also showed that when human DPCs and human follicular stem cells (FSCs) were mixed and cultured on Matrigel for several days, the structure of hair-like can be found. Genetic evidence suggests crucial roles for PDGF, TGF-b, VEGF, IGF-1 and FGF signaling in HF development and cycling. Platelet-rich plasma (PRP) is an autologous preparation of platelets in concentrated plasma, and containing various growth factors. Thus, PRP has been used in a wide variety of surgical procedures and clinical treatments. Our previous study showed a considerable effect of PRP on the time of hair formation and the yield of HF reconstitution. Based on the previous studies, we hypothesis that Matrigel and PRP can be used to constructed a biomimetic cell microenvironment, which in turn benefit to the construction of bioengineered HF from dissociated adult human DPCs and FSCs. Thus, the aim of this study is to construct and evaluate bioengineered HF from dissociated adult human DPCs, FSCs, Matrigel and PRP.

毛囊再生是秃发治疗的终极选择，因伦理限制和物种差异，既往研究多局限于动物的体内实验。虽然目前尚未在体外构建出人组织工程毛囊，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处于丰富细胞外基质的包裹之中，从EHS肿瘤中提取的Matrigel是一种拟生态细胞外基质，被广泛用于细胞的三维培养。我们在前期研究中首次发现将人毛乳头细胞和毛囊干细胞接种于Matrigel进行三维培养时可观察到毛发纤维的形成。以往研究还发现含有多种生长因子的PRP有助于增强毛乳头细胞的增殖和诱导功能。由此我们拟采用Matrigel和PRP构建出适合毛囊发育的仿生细胞微环境，通过人毛乳头细胞和毛囊干细胞的三维培养而构建组织工程毛囊。不仅可为毛囊发育、毛囊相关疾病的发病机制和毛发疾病治疗药物的筛选研究提供实验模型，也可为治疗秃发治疗和构建具有附属器的组织工程皮肤提供研究基础。

秃发是一种常见的毛发相关性疾病，无论是药物治疗还是自体毛发移植手术，均不能再生出新的毛囊。随着组织工程与再生医学的发展，构建组织工程毛囊以实现毛囊再生将会是治疗秃发较有前景的解决方案。目前，人源性毛囊重建目前尚未实现，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（DPC）处于丰富细胞外基质的包裹之中，从EHS肿瘤中提取的Matrigel 是一种拟生态细胞外基质，被广泛用于细胞的三维培养。以往研究还发现含有多种生长因子的PRP有助于增强DPCs的增殖和诱导功能。研究中，我们试图采用Matrigel和PRP构建出适合毛囊发育的仿生细胞微环境，通过人DPCs和毛囊干细胞（FSCs）的三维培养而构建组织工程毛囊。经过一系列研究，首先，我们实现了人FSCs和DPCs的分离、培养，并根据国内外研究热点，我们还探讨了基质金属蛋白酶及其抑制剂与毛囊周期的相关性。其次，我们通过Matrigel 、PRP、层层自组装（LBL）、悬滴法等方法，为毛囊再生探索适宜的仿生微环境，并构建组织工程毛囊。最后将FSCs和DPCs以一定比例在体外和体内实现毛囊重建及再生，还探讨了雄激素脱发的分子学机制。以上研究为毛囊组织工程的进一步研究提供了一定理论和研究基础。