单细胞测序lncRNA对天祝白牦牛毛囊周期性生长的调控机制研究

Yak hair is producted by secondary hair follicle(SF), is a rare and special high-grade textile material. Hair follicle cycling is an important physiological characteristic for yak to adapt to the Tibetan plateau environmental changes.However,the precise molecular mechanism of yak hair follicle cycling remains elusive.Several studies have suggested that hair follicle cycling is an orchestrated interaction between mesenchymal and epithelial cells mediated through a series of ongoing and complex signaling pathways. Therefore, in order to elucidate the precise regulatory mechanisms of long non-coding RNAs in Yak secondary hair follicle cycling,firstly,specifically high purity dermal,epidermal,dermal papilla single cells will be separated from Tianzhu yak midside skin strips through laser capture microdissection at catagen,telogen and anagen stages in the hair cycle.Then, the special lncRNA and its target mRNA in secondary follicle induction will be screened and predictived by using of "joint analysis of lncRNA and mRNA expression profiles " by single-cell strand-specific RNA sequencing and validated by luciferase reporter system. Finally, the function of miRNA,lncRNA will be detected by using CRISPR-Cas9.The study results will provide the new ideas for the study of molecular mechanisms of adaptation to environmental changes at Qinghai-Tibetan Plateau, moreover, provide a reliable scientific basis for improving the yak hair quality.

牦牛绒是由牦牛次级毛囊（SF, secondary hair follicle）产生的，是一种稀有的特种高档毛纺原料。牦牛绒呈周期性生长是其适应青藏高原环境变化的重要生理特征，但尚未见有关牦牛SF周期性生长调控机制的研究报道。研究表明，人和鼠等模式动物毛囊周期性生长涉及一系列复杂的表皮、真皮和毛囊间持续而复杂的信号通路。因此，本研究在应用激光显微切割获得天祝白牦牛SF周期性生长阶段表皮、真皮和SF毛乳头单细胞的基础上，进行单细胞链特异性RNA-seq，识别牦牛SF周期性生长阶段与靶mRNA互作的lncRNA，筛选特异调控牦牛SF周期性生长的lncRNA和功能基因，以期解析lncRNA对天祝白牦牛毛囊周期性生长的调控机制，有助于解析牦牛对青藏高原高寒环境适应的分子机制，为提高牦牛绒品质提供技术参考与理论依据。

牦牛毛绒呈周期性生长已成为牦牛适应青藏高原高寒环境变化的重要的生理特征。为解析牦牛毛囊周期性生长的调控机制，本研究以天祝白牦牛为研究对象，首先开展了牦牛毛囊周期性生长形态特征分析，确定了牦牛毛囊周期性生长（生长期、退行期和休止期）的时间节点：10 ~ 1 月份为生长期， 2 ~ 6 月份为退行期，7 ~ 9 月份为休止期；通过测定不同毛囊周期性生长阶段的重要体内激素含量，发现催乳素、睾酮、雌二醇、甲状腺素、褪黑素、胰岛素样生长因子2和生长激素参与毛囊周期性生长调控。根据确定的牦牛 HFs周期性生长的时间节点，分别应用常规皮肤转录组、单细胞转录组（激光显微切割、10×Genomics单细胞测序）测序开展了牦牛毛囊周期性生长表达谱研究，共筛选到2316基因、2884个lncRNA参与毛囊周期性生长，并显著富集到Wnt信号通路、VEGF信号通路和调控干细胞多能性的信号、FGF信号通路、Hippo信号通路、MTOR和Hedgehog信号通路，建立了毛囊周期性生长关键信号因子的表达模式和调控网络图；利用实时荧光定量PCR、免疫组化技术研究了Wnt10b、FGF18在毛囊周期性生长过程中的表达规律，研究表明Wnt10b 促进毛囊生长，而FGF18抑制毛囊活性使毛囊进入休止期。上述研究实际上首次系统研究了牦牛毛囊周期性生长形态特征、关键时间点和转录组表达谱，筛选到Wnt信号通路、VEGF信号通路、FGF信号通路等通路的18个关键基因参与毛囊周期性生长分子调控网络，为有助于解析牦牛对青藏高原高寒环境的适应的分子机制，为提高牦牛绒品质提供可靠的技术参考与理论依据。