【作者】 席海燕；

【导师】 周欢敏；

【作者基本信息】 内蒙古农业大学 ， 动物发育生物学与生物技术， 2009， 博士

【摘要】 本论文旨在研究绒山羊皮肤干细胞的分布位置及迁移轨迹,构建生长期和休止期次级毛囊差异表达基因文库,筛选与绒山羊次级毛囊生长相关的特异表达基因,从而揭示产绒动物不同类型的毛囊干细胞的行为特征、定位分布、相互关系,特别是尚未见报道的次级毛囊的干细胞生物学特性,为建立毛囊干细胞休眠、增殖、迁移、分化和决定等生命活动过程和机制的模式提供方法和理论依据。选择体态相近、健康的周岁绒山羊,分成两组:标记BrdU的实验组和未标记BrdU的对照组。根据干细胞的慢周期特性,对绒山羊表皮细胞DNA进行BrdU标记,定期取背部皮样,4%多聚甲醛固定,常规石蜡包埋切片,免疫组化检测,结果前10周内BrdU结果呈阳性。8周,BrdU标记细胞主要位于初级、次级毛囊的毛球部,毛干也有分布;8~10周,BrdU标记细胞仅位于初、次级毛囊的外根鞘;10周后无阳性信号分布。选择BrdU标记8周和8~10周的绒山羊背部皮样,分离酶II 4℃消化3小时,分离完整毛囊。分别用分段培养法和IV型胶原粘附法对毛囊标记细胞(毛球部和毛囊中段)进行培养,能形成典型的干细胞克隆,且细胞直径较小,核质比较大,符合皮肤干细胞的形态学特征。用皮肤干细胞的标志分子P63对BrdU标记细胞进行免疫组化鉴定,发现BrdU标记滞留细胞呈阳性。以上结果证明BrdU标记滞留细胞即为皮肤干细胞,初步确定干细胞位于初级、次级毛囊的毛母质和外根鞘,在表皮基底层也有分布。为进一步阐明次级毛囊周期性重建过程中干细胞分化启动-休眠的调控基因,以生长期和休止期次级毛囊为研究对象,利用抑制性消减杂交技术,构建了具有高消减效率的生长和休止期次级毛囊的cDNA文库。挑取20个阳性克隆,用Nested PCR Primer 1和2R进行PCR扩增,插入片段长度主要分布在250~1000 bp之间。随机挑取正、反向两个差异表达基因文库中的单克隆各750个进行PCR扩增、测序,分别获得309条和344条有效序列,平均长度分别为693bp和596bp。将其进行同源性分析,各获得224个和300个nucleotide数据库中山羊及其他物种的已知存在序列同源。在EST数据库中分别找到76个、38个相似EST序列,还有9个和6个无同源性序列。在GenBank数据库共注册非冗余ESTs 126条,序列号为64970025~64970150。对已知功能基因的ESTs进行分类,正反库中细胞分裂类各为15个和13个、细胞信号类为44个和49个、细胞结构蛋白30个和52个、细胞防御类13个和21个、代谢类24个和46个、基因/蛋白表达类33个和37个、未分类的65个和82个。在此基础上,进行了两次荧光交换的cDNA芯片杂交,以筛选差异表达基因。发现6个次级毛囊生长期的差异基因,其中上调基因3个,下调基因3个;10个次级毛囊休止期的差异基因,其中上调基因9个,下调基因1个。总之,本研究首次用BrdU活体标记追踪的方法证明绒山羊初级、次级毛囊及表皮中均存在皮肤干细胞;首次建立了用SSH方法分离绒山羊次级毛囊生长和休止期差异表达基因的方法,并结合荧光交换的cDNA芯片杂交来筛选。所发现的差异表达基因对阐明绒山羊毛囊干细胞休眠维持和分化启动的可能分子机制、探索干细胞技术在将来动物分子育种和绒毛生产中应用的可能性提供方法和理论依据,但对这些差异表达的基因尤其是功能未知的基因在绒毛生长中的确切生物学功能仍需进一步深入研究。更多还原

【Abstract】 The research was carried out to study the localization and migration of skin stem cells, to construct suppression subtractive hybridization(SSH) libraries of secondary follicles in anagen and to search some candidate genes involved in cashmere development in Inner Mongolia cashmere goat with cDNA micrarray. The information generated in this study can reveal behavior characteristic and orientation distribution of follicle stem cells, especially biological characteristics of secondary follicle stem cells, also can privide theoretical basis and method on buliding mechanism model of dormancy, migration, proliferation, differentiation, decision processes of follicle stem cells.The healthy cashmere goats were divided into two groups: the test group labeled BrdU and the control group unlabeled BrdU.BrdU was injected intravenously for a total of two injections. According to skin stem cells characteristics of the slow cycle, skin samples were took from the back skin of the goats,fixed in 4% paraformaldehyde, embedded with paraffin and sectioned,detected by immunohistochemistry.BrdU could be detected only 10 weeks in Inner Mongolia cashmere goats and could be used for large animals in vivo short-term markers. Intraperitoneal injection 8 weeks, positive signals located mainly in hair bulb of primary and sencondary follicle,also in hair shaft. Intraperitoneal injection 8-10 weeks, positive signals obvirously decreased,only could be found in outer root sheath. After 10 weeks, almost no positive signals distribution. The back skin samples labeled BrdU for 8 and 10 weeks were incubated in 0.2% dispaseII at 4℃for 3h,isolated primary and secondary follicles under a dissecting microscope,then cultured label-retaining cells (LRC) of hair follicles(hair bulb and follicle midpiece) in vitro with segmentation culture method and collagen IV adhesion culture method respectively.The LRCs showed a large nuclear/cytoplasmic ratio and small size,and clonogenicity with the morphological characteristics of skin stem cells.To evaluate the BrdU LRCs, P63, a possible epidermal stem cell specific marker, was used. Immunohistochemical results idicated BrdU LRC were positive for P63. These results showed that BrdU LRCs were epidermal stem cells.The stem cells possiblly located in not only hair matrix and outer root sheath of primary,secondary follicle,but also the epidermal basal layer.To further clarify the startup–dormancy control genes of stem cells during secondary follicle reconstruction periodically,forward-SSH library was performed with cDNA from secondary follicle in anagen as the“tester”and cDNA from secondary follicle in telogen as the“driver”. While reverse-SSH library was performed with cDNA from secondary follicle in telogen as the“tester”and cDNA from secondary follicle in anagen as the“driver”.The gene fragments were sequenced, and analyzed by bioinformatics. Two SSH libraries had been constructed successfully. Twenty positive clones were amplificated by using nested PCR primer 1 and 2R, the size of inserts was 250-1000bp.309 genes and 344 genes were obtained respectively by DNA sequencing 750 clones picked randomly from two SSH libraries. Their average length were 693bp and 596bp.After nucleotide blast homological analysis,224 matched to known genes, seventy-six matched only to other ESTs in dbEST, and the remaining nine showed no match to any ESTs or known genes in forward-SSH library. While 300 matched to known genes, thirty-eight matched only to other ESTs in dbEST, and the remaining six showed no match to any ESTs or known genes in reverse-SSH library. 126 redundancy ESTs were registered in the GenBank,their accession numbers were 64970025~ 64970150. These were categorized into seven categories on the basis of gene function. They were divided into cell division(15,13), cell signaling/communication(44, 49),cell structure/motility(30,52),cell organism defense(13,21),metabolism(24,46),gene/protein expression(33,37) and unclassed (65,82).On this basis, the exchange of two fluorescent hybridizations of the cDNA chip were used to screen specific differentially expressed genes. A total of six differentially expressed genes were found in anagen including three up-regulated genes and three down-regulated genes.While ten differentially expressed genes were found in telogen including nine up-regulated genes and one down-regulated gene.In conclusion,it is the first time that stem cells are located in primary, secondary follicles and epidermis of cashmere goats by BrdU labeling method;it is the first time that differentially expressed genes in secondary follicles of anagen and telogen are isolated by SSH,combined with the exchange of fluorescent cDNA chip hybridization to screen differentially expressed genes.The results are useful for elucidating the possible molecular mechanism on keeping dormancy and starting differentiation by stem cells, but it is also necessary to elucidate biological functions of differentially expressed genes,especially genes with unknown functions.更多还原