Wnt10b基因对安哥拉长毛兔次级毛囊周期的表观遗传调控机制研究

Although rabbit hair was high-grade natural materials of textile, the development of Angora rabbit industry was restricted by its low production. Cashmere was fibers from secondary hair follicle, the production and quality were affected directly by cycle of hair. follicle. However, because it was not clear in the mechanism regulating hair follicle cycle, improving the quality of hair from genetic is difficult. Wnt10b gene might be involved in the development process of Angora rabbit hair fiber, according to the results of Agilent’s Rabbit expression profile chip, which screened differentially expressed genes in two varieties of rabbits’ hair density(New Zealand white rabbit and Angora rabbit). Studies in other species indicated that Wnt10b may be involved in cycles of hair follicles by activation of Wnt/β-catenin pathway. In present study, Q-PCR, tissue staining, BSP and CHIP were used, cycle of secondary hair follicle of Angora rabbit were observed in vivo, the main studies were as following: ①definition of anagen, catagen and telogen 3 phases during secondary hair follicle cycle; ②regulation of Wnt10b gene of secondary hair follicle cycle; ③epigenetic regulation of Wnt10b gene of secondary hair follicle cycle. In order to clarify regulation of Wnt10b gene in cycle of secondary hair follicle, reveal the mechanism of follicle cycle, elucidate the influence of genetic factors on yield and quality of rabbit hair, and promote the process of molecular breeding of high-production and high-quality Angora rabbit.

安哥拉兔兔绒是高档纺织原料，但因其产量较低，严重制约了长毛兔产业的发展。绒是皮肤次级毛囊生长的纤维，毛囊周期性变化直接影响其产量。目前家兔次级毛囊周期性循环的调控机制不清，无法从遗传水平改善兔绒品质。家兔全基因组表达芯片证实Wnt10b同毛纤维生长发育相关。其他物种研究表明Wnt10b可激活Wnt/β-catenin途径而参与毛囊周期性循环。因此，本项目以次级毛囊周期为研究对象，组织切片染色鉴定其生长期、衰退期及休止期；Q-PCR检测Wnt10b基因的表达规律；PCR产物直接测序分析Wnt10b启动子多态性；重亚硫酸氢盐测序和染色质免疫共沉淀分析启动子DNA甲基化和组蛋白乙酰化水平。最终揭示次级毛囊周期中Wnt10b基因转录调控的表观修饰机制，阐明Wnt10b基因对次级毛囊周期的调控机制，将对解析家兔毛囊发育的分子调控机制，加快高产、优质绒毛长毛兔的分子育种新技术进程有重要意义。

长毛兔兔绒具有保暖、轻盈、吸湿性好等优点，是天然的高档纺织品原料，但因其产量较低，质量参差不齐，严重削弱了其市场竞争力。次级毛囊作为毛纤维的“发源地”，是影响绒产量和品质的起点。目前毛兔次级毛囊周期性循环的调控机制尚不清楚，所以无法从遗传水平改善兔绒品质。全基因组表达芯片证实Wnt10b同兔毛纤维生长发育相关，其他物种研究表明Wnt10b可激活Wnt/β-catenin途径而参与毛囊周期性循环。本研究以次级毛囊周期为研究对象：（1）采用HE染色技术分析不同月龄毛兔皮肤组织切片，结合Q-PCR技术分析生长期和衰退期Marker基因LEF1和TGFβ1，判定7.5月龄为毛囊生长期，5月龄为衰退期，6月龄为休止期。确定了长毛兔的次级毛囊周期。（2）Q-PCR检测次级毛囊周期中Wnt10b基因的表达规律，生长期毛囊中Wnt10b水平显著高于衰退期和休止期，表明Wnt10b在调控毛兔的次级毛囊周期中有重要作用；体外研究也证实，Wntl0b可通过经典Wnt途径作用于毛兔毛乳头细胞，激活β-catenin的表达，上调与毛囊诱导能力相关的LEF1、IGF1和ALP的表达水平，促进毛乳头细胞的增殖，成功维持长期培养后毛兔毛乳头细胞的毛囊诱导能力；PCR产物双向测序证实，Wnt10b外显子1，4和5，以及上游2000bp启动子在306个家兔均不存在多态性位点。（3）重亚硫酸氢盐测序分析证实，启动子CpG岛-175位点的DNA甲基化水平同Wnt10b基因mRNA表达量呈极显著负相关，该位点甲基化水平增高会抑制Wnt10b基因的表达；染色质免疫共沉淀分析显示，-256～-11bp和98～361bp两个区段的组蛋白H3乙酰化水平影响Wnt10b基因的表达，且乙酰化水平增高会促进Wnt10b基因的表达；而第-175位点的甲基化可能同-256～-11bp区段的组蛋白H3乙酰化协同作用，共同调控次级毛囊周期Wnt10b基因mRNA的表达。总之，本项目通过对毛兔次级毛囊周期的Wnt10b基因的表达规律、启动子CpG岛DNA甲基化及组蛋白H3乙酰化水平，基因多态性及体外Wnt10b参与Wnt信号途径的调控机制进行研究，阐明了Wnt10b基因对次级毛囊周期的调控机制，将对解析家兔毛囊发育的分子调控机制，加快高产、优质绒毛长毛兔的分子育种新技术进程有重要意义。

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