lncRNAs调控绒山羊精原干细胞体外诱导分化的分子机制

Spermatogonial stem cells (SSCs) are the only adult stem cells during the process of mammalian spermatogenesis. The differentiation of SSCs is regulated by the epigenetic mechanism which mediated by long non-coding RNAs (lncRNAs). Until now, the epigenetic mechanism of lncRNAs regulatingthe process of SSCs differentiation in vitro is elusive. In this study, the SSCs of Shanbei white cashmere goats are isolated by the immunomagnetic microbeads combining with different plating methods. The culturing system of SSCs will be optimized after purifying the collected SSCs. The specific lncRNAs involved in SSCs differentiation are screened by lncRNA sequencing (lncRNA-seq) technology. The target genes of specific lncRNAs in different differentiation stages will be verified by RNA immunprecipitation and Dual-Luciferase Reporter Assay. The function of specific lncRNAs is verified by constructing over-expression vector and interference vector. The methylation mode of target genes expression will be constructed. The epigenetic regulation mechanism of methylase and demethylase on the target genes of lncRNAs was investigated by constructing over-expression vector and interference vector of methylase and demethylase. In this study, the molecular mechanisms of lncRNAs on induced differentiation of cashmere goats SSCs will be revealed. This study will benefit the understanding of mechanisms in male mammalian spermatogenesis.

精原干细胞（SSCs）是维持动物精子发生的唯一成体干细胞，长链非编码RNAs（lncRNAs）介导的表观遗传调控机制在其分化过程中发挥关键作用，但关于lncRNAs调控山羊SSCs诱导分化的表观遗传机制尚不明确。本项目以陕北白绒山羊为研究对象，用免疫磁珠法结合差异贴壁法分离羔羊SSCs，纯化后优化培养体系；通过维甲酸（RA）诱导获得单倍体生殖细胞，利用lncRNA-seq技术获得SSCs诱导分化前后的特异lncRNAs；双荧光素酶报告系统和RNA免疫沉淀技术验证lncRNAs的靶基因，构建超表达和干扰载体，对其功能进行验证；构建特异lncRNAs靶基因的甲基化表达模式，对其调控的靶基因进行甲基化研究；构建甲基化酶和去甲基化酶超表达和干扰载体，研究甲基化酶和去甲基化酶对靶基因的表观遗传调控作用，揭示lncRNAs调控绒山羊SSCs诱导分化的分子机制，为研究其他动物精子发生机理奠定基础。

稳定的精子发生依赖于精原干细胞(SSCs)的自我更新与定向分化，长链非编码RNAs（lncRNAs）在其分化过程中发挥关键作用。但是，关于山羊SSCs的体外培养、诱导分化以及lncRNAs调控山羊SSCs诱导分化的研究非常有限。.项目组取得了如下研究成果：.（1）使用不同发育阶段山羊睾丸组织，探究睾丸精原细胞动态增殖分化特征。结果表明，未分化精原细胞占比在75日龄前最高。添加25 ng/mL的EGF和20 ng/mL的PDGF-BB体外培养28 d后，克隆团为THY1和PLZF阳性，表明克隆团为未分化的精原细胞，成功建立了山羊SSCs体外长期培养体系。.（2）探究褪黑素在SSCs冷冻保存过程中提高冷冻保存效果的机制。结果表明，10-6 mol/L褪黑素可提高细胞抗氧化和抗凋亡能力，同时减少细胞自噬。利用维甲酸（RA）和骨形态生成蛋白4（BMP4）构建SSCs体外分化体系，10 nM RA 和50 ng/mL BMP4均有利于促进SSCs的分化，BMP4通过Smad、PI3K信号途径发挥作用。.（3）对45 d和240 d的山羊曲细精管组织进行全转录组测序，鉴定出11612个lncRNA。发现GSKIP、KAT8、MBD3L1等是lncRNA tcon_00029023、tcon_00035144、tcon_00017872、tcon_00051085和tcon_00033882的顺式或反式的靶基因。构建LncRNATCONS-00051058超表达载体发现，腺病毒和慢病毒包装的LncRNA都不能够转染进SSCs，但能够转染支持细胞。.（4）收集曲细精管中的25373个细胞进行单细胞转录组测序，结果显示，SOHLH1、DMRT1、YBX2、HSPB9、MISP3、LYPD4、PRSS37和SPEM1分别在精原细胞、早期精母细胞、精母细胞和精子细胞中特异表达。INHA、EGR1、INSL3、ACTA2、LOC100860813、LOC102189356、SOX18和TM4SF1分别在不同的微环境细胞中表达，建立了山羊精子发生转录图谱。.本项目研究结果对于探究山羊SSCs体外培养、诱导分化以及冷冻保存具有重要意义，也将为揭示山羊精子发生的细胞命运转录机制提供了理论参考。