毛乳头细胞仿生微环境三维培养体系的构建及其生物学调控作用

Dermal papilla cells(DPCs) are vital seed cells in bioengineering for hair follicle regeneration. However, in in-vitro culture, the lack of microenvironment inevitably led to the inductivity loss of DPCs. In order to obtain a large number of DPCs with inductivity, several culture methods have been developed. Nonetheless, neither of these methods can fully restore the characteristics of DPCs. It is demonstrated that in the hair follicle, extracellular matrix, dermal sheath cells and hair germinal matrix cells together enclose the DPCs, forming the microenvironment which play a significant role in the regulation of DPCs' characteristics. Deprivation of the microenvironment in previous culture methods is the key factor contributing to the DPCs incapacitation. In our preliminary research, we set up a bionic three-dimensional culture in which DPCs were co-cultured with hair germinal matrix cells in Matrigel gel. Though still short on the time of DPC inductivity maintenance, this culture significantly enhanced the biological characteristics of DPCs. Recent studies indicated that dermal sheath cells(DSCs) have a valuable function in maintaining the biological characteristics of DPCs. Based on the mentioned above, from the bionic perspective, we intend to establish a three-dimensional culture system of DPCs based on the 3D microenvironment of Matrigel, DSCs and hair germinal matrix cells, simulating the in vivo microenvironment of DPCs and thus constructing the best 3D culture system. Meanwhile, the High-throughput Transcriptome Sequencing will be used to screen the key genes which regulate the function of DPCs through DSCs. In this way, we will further elucidate the specific mechanism of microenvironment in regulating the function of DPCs.

作为毛囊组织工程的核心种子细胞，毛乳头细胞（Dermal papilla cells，DPCs）在体外培养过程中会逐渐丧失生物学功能。既往培养方法忽略了体内结构中DPCs周围的细胞外基质，毛母质细胞和真皮鞘细胞所组成的微环境对其生物学功能的影响。前期我们从仿生微环境角度出发构建了基于基质胶的DPCs与毛母质细胞的共培养体系，发现其相比其它体系，能更好地维持DPCs的生物学功能，但在维持时间上仍有欠缺。最新研究证实真皮鞘细胞对于DPCs生物学功能的维持至关重要。因此，本项目拟从细胞微环境、非细胞成分骨架和空间结构等角度模拟DPCs体内微环境，借助于真皮鞘细胞、毛母质细胞和基质胶构建出DPCs的体外仿生微环境三维培养体系。同时结合高通量测序转录组学技术筛选出真皮鞘细胞介导微环境调控DPCs生物学功能的重要基因，并进一步探讨其调控机制，最终为组织工程毛囊和功能性组织工程皮肤的大量构建奠定基础。

大面积秃发在世界范围内变得越来越普遍，无论是药物治疗还是自体毛发移植，均因受限无法取得满意疗效。随着组织工程和仿生技术的不断完善，通过真皮乳头细胞（DPCs）构建仿生化毛囊可为大面积脱发的治疗提供解决方案。研究表明三维培养有助于DPCs形成体内真皮乳头样结构。本课题首先建立了Matrigel（一种小鼠EHS肿瘤提取的拟生态细胞外基质，含丰富蛋白及多种生长因子）包裹DPCs三维培养模型——DPCs聚集物（DPAs）模型，其毛发相关基因的维持优于传统三维培养方式。然后在此基础上依据DPCs体内微环境进一步仿生化，联合真皮鞘杯细胞（DSCCs）、毛母质细胞（HMCs）构建出仿生共培养体系。最后，我们验证了该体系规模化标准化制备的可行性。研究表明，并该体系具有适合毛囊发育的仿生细胞微环境，较传统方式更高效地诱导出组织工程毛囊，并具有规模制备的优势。在此过程中，根据国内外研究热点结合试验所需，我们还规范了FUE治疗大面积秃发临床应用技术，同时建立了人胎盘制作生物水凝胶培养DPCs模型，深入研究了PRP、生长因子对DPCs诱导能力的影响，还揭示了外泌体作为毛囊细胞间交流重要影响及机制。以上一系列研究不仅可为毛囊细胞间信号转导、毛囊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，也为毛囊组织工程的进一步研究和大规模秃发治疗提供了重要的理论和研究基础。