【作者】 吴江鸿；

【导师】 闫祖威；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 绒山羊是重要的毛用经济动物,其绒毛的生长发育受多种因素影响。Hoxc13已被证实可以直接调控两类毛发组成蛋白(角蛋白与角蛋白关联蛋白)的表达,且Hoxc13功能的获得和缺失都会形成脱毛型小鼠。本论文主要研究了绒山羊毛囊发育不同阶段,皮肤中Hoxc13的表达规律;同时研究了过表达Hoxc13基因对体外培养的皮肤细胞中HFDRGs的影响;另外通过分析埋植/添加褪黑激素与对照组之间HFDRGs的表达变化情况,了解褪黑激素对HFDRGs的作用。本研究共得到以下几点结论:1、本研究克隆了绒山羊Hoxc13基因,序列长7.2 kb,包含2个外显子、1个内含子,基因登录号EU839660,CDS长990 bp,共编码330个氨基酸。通过分析3个纲17个目的28个脊椎动物Hoxc13基因及其推定的氨基酸序列,发现Hoxc13基因在非哺乳动物向哺乳动物进化过程中出现了1次基因插入,插入序列为富含甘氨酸的低复杂区域(Low-Complexity Region,LCR),该区域的长度在哺乳动物中发生了分化(10-28个氨基酸的变异)。哺乳动物Hoxc13基因出现了适应性进化,可能与被毛的起源有关。2、绒山羊皮肤中Hoxc13基因的表达量与胎儿时期皮肤厚度的变化趋势相一致;在成年绒山羊皮肤中,Hoxc13基因的表达量与次级毛囊活性周期显著相关(R=0.837, p<0.001);埋植褪黑激素可以提前绒山羊皮肤中Hoxc13的表达,且显著上调8-10月份皮肤中Hoxc13的表达量。在体外,褪黑激素对绒山羊皮肤细胞中Hoxc13基因的诱导表达效果不同,下调了成纤维细胞中Hoxc13的表达,却上调了角质化细胞中Hoxc13的表达量。3、过表达Hoxc13基因可能导致角质化细胞分化死亡,未能获得稳定表达Hoxc13基因的角质化细胞克隆,但获得了稳定表达Hoxc13基因的成纤维细胞系;皮肤成纤维细胞过表达Hoxc13,可促进TgfβrII、Rorα2、Nanog、Wnt10b和PdgfrA的表达,抑制Bmp2、Msx2、Ntrk3和Detal基因的表达。总之,Hoxc13基因在哺乳动物被毛的起源过程中发生了适应性进化,体内外研究表明Hoxc13在绒山羊皮肤毛囊形态发生、周期性生长过程中扮演重要角色。该论文为今后进一步研究绒山羊及其他毛用动物的毛囊发育机理奠定了理论基础。更多还原

【Abstract】 Cashmere goat is a very important economic animals provided wool and cashmere. Coat hair growth can be affected by many factors. Hoxc13 has an important role in controlling hair formation through regulating keratin differentiation-specific genes. Hoxc13 deficient and overexpressing mice exhibit severe hair growth and patterning defects. Therefore, we detected the expression level of Hoxc13 in cashmere goat skin from different developmental phases of hair follicle; and investigated the effect and interaction relations for over-expression Hoxc13 and other HFDRGs of skin cells in vitro; and clarified the role of melatonin with relation to HFDRGs by analyzing the changes of HFDRGs expressing between melatonin implant/add and control.The following conclusions were obtained from the study.1. In this study, the Hoxc13 gene of cashmere goat was cloned (Assession number: EU839660), The gene spans 7.2 kilobases (kb) and has two exon which seperated by a relatively large intron, which has a coding sequence of 990 nucleotides that putative 330 amino acids. By analyzing the protein sequence of Hoxc13 in 28 species of vertebrates which belonged to 3 classes and 17 orders, we found that an insertion of low complexity region(LCR) in N teminal of Hoxc13 during the divergence of the therapsid lineage (leading to mammals) from the sauropsid lineage (reptiles, birds). The divergence of length of the LCR occurs in mammal(10-28Aa). Our results implicate adaptive evolution of Hoxc13 genes occurs in the origin and diversification of the mammals.2. The changes of the Hoxc13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis. In adult goat, the expression level of Hoxc13 in skin associated with the follicle activity. Melatonin may get to expression cycle of Hoxc13 in cashmere goat skin in advance, and the expression level of the Hoxc13 was significantly increased during 8-10 month. In vitro, melatonin has a diversity effect to fibroblasts and keratinocytes. It could increase the expression level of Hoxc13 in keratinocytes, while inhibit the Hoxc13 expression in fibroblasts.3. Overexpression Hoxc13 in keratinocytes could result in differentiation or die. Thus, we did not obtain the cell line that stablily expressed Hoxc13. However, a fibroblast cell lines with stable gene expression Hoxc13 was established. Overexpression Hoxc13 in fibroblasts of cashmere goat skin up-regulated TgfβrII, Rorα2, Nanog, Wnt10b and PdgfrA, and down-regulated Bmp2, Msx2, Ntrk3 and Detal.In summary, adaptive evolution of Hoxc13 gene occurred in the origin and diversification of the mammalian coat hair. And Hoxc13 played an impotant role in goat skin and hair follicle morphogenesis/cycle by in vivo and in vitro researching. This study lay a fundation to further research on cashmere goats and other wool animals for developing mechanism of the hair follicle.更多还原