绒山羊毛乳头细胞外分泌蛋白诱导毛母质细胞增殖的分子机制研究

Skin hair follicle (HF) in cashmere goats is an effective organ for production of hair fibers, and over 20 different types of cells are involved. Of them, the dermal papilla cells (DPCs) and hair matrix cells (HMCs) are two key components that play pivotal roles in hair renewal and periodic cycling of HFs. Elucidation the underlying mechanism regarding to the secreted proteins (EPs) by DPCs to HMCs is key for artificial manipulation of hair (cashmere) fiber growth and molecular breeding for the cashmere goat industry. ..We have previously established the cell culture systems for multiple cell types (DPCs, HMCs, hair stem cells, etc) in HFs from cashmere goat skins. In this project, we are going to use the techniques including cell co-culture, RNA-seq, and iTRAQ, to identify the EPs that are important for the proliferation of HMCs. Next, the CCK8, FACS will be used to determine the effects of adding artificial recombination protein and inhibiting key genes within the pathways for the proliferation of HMCs. Subsequently, the CRIPSR/Cas9 system will be used to disrupt key receptors of EPs. Collectively, we will depict the interaction between DPCs and HMCs within HFs of goat skins. Our results will illustrate the molecular mechanism that the EPs involved for the proliferation of HMCs, and further benefit the improvement of molecular breeding programs in cashmere goats.

绒山羊皮肤毛囊是一个高效的生发器官，由20多种细胞构成，其中毛乳头细胞和毛母质细胞是其最重要成员，在毛发再生和毛囊周期循环中有着重要作用。探明毛乳头细胞分泌的关键蛋白对毛母质细胞增殖的作用机制，对实现羊绒再生调控，促进绒山羊分子育种具有重要意义。在成功实现绒山羊毛囊多种细胞体外培养的基础上，本项目进一步运用细胞共培养、转录组学和蛋白质组学方法，鉴定由毛乳头细胞分泌的对毛母质细胞增殖起关键作用的蛋白；之后，运用CCK8、流式细胞分选等技术研究添加人工重组外分泌蛋白、及利用信号通路抑制剂阻断细胞增殖信号通路的关键节点对毛母质细胞增殖的影响；最后，利用CRISPR/Cas9技术在毛母质细胞模型中敲除关键外分泌蛋白的受体基因，验证其对毛母质细胞增殖的影响。综合以上结果，构建毛乳头细胞与毛母质细胞之间信号网络互作关系图，揭示毛乳头细胞通过分泌关键蛋白诱导毛母质细胞增殖和对毛囊周期循环调控的分子机理。

毛乳头细胞（DPs）所分泌的外泌体能够起到逆转毛囊微型化的作用，使缩小的毛囊重新变大。阐明绒山羊毛乳头细胞外泌体对毛囊周期性发育的分子调控机理，为实现人工调控绒毛生长、加快遗传育种进展奠定理论基础。本项目的主要研究内容包括：（1）通过Transwell小室介导的毛乳头细胞和毛母质细胞共培养方法，建立并优化毛乳头细胞和毛母质细胞体外共培养体系；（2）利用RNA-seq技术探索毛乳头细胞和毛母质细胞的共表达条件下的表达模式，发现共培养条件下毛乳头细胞上调基因3358个，包括已知功能基因，如FGF7、FGF10、WNT5A和IL-6等；毛母质细胞上调基因3436个，包括诱导毛乳头细胞表达生长因子的基因IL-1A等，下调基因3059个，包括编码角蛋白的KRT17等和对角蛋白表达起调控作用的HOXC13等；（3）分离得到DPCs来源的外泌体并证明了DPCs来源的外泌体可以运输到HFSCs，且能被HFSCs摄取。对外泌体miRNA测序分析发现111个miRNA在DPC-Exos和DPCs中差异表达。（4）对外泌体miR-22-5p与let-7b-5p构建候选靶基因的双荧光素酶报告载体，阐明其对HFSCs的调控途径和验证相关靶基因（LEF1、FOXC1和MSI2）。此外本项目还做了进一步拓展研究：ATRA、瘦素对毛乳头细胞和毛囊发育的影响机制的分析；褪黑素对绒山羊毛乳头细胞增殖和凋亡的影响以及Sirt7基因过表达对绒山羊毛囊发育的调控研究。本项目结果为提高绒山羊产绒性能的分子育种提供候选基因，为进一步阐述miRNAs、lncRNAs、circRNAs在毛囊发育中的功能和分子机制奠定了基础，同时有助于揭示外泌体在毛囊干细胞周期性发育过程中分子调控机制。