【作者】 刘迎春；

【导师】 周欢敏；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 本研究旨在建立绒山羊皮肤干细胞的分离、纯化培养体系,并对其特性和分化潜能进行进一步探讨,以便为今后利用皮肤干细胞进行绒山羊毛囊的定向调控提供科学的理论依据。选择健康成年绒山羊背部皮样,经中性蛋白酶4℃消化过夜,分离表皮和毛囊,用胰酶消化后解离细胞,再用IV型胶原粘附法对消化细胞进行筛选分离,经IV型胶原粘附10min的绒山羊皮肤干细胞直径较小,核质比较大,培养过程中能够形成鸟巢状、岛状和铺路石状等不同形状的细胞克隆,而且在传代的过程中,大多数细胞始终维持在G1/G0期,各代克隆形成率也比较高,部分细胞表达K15、K19、p63、CD34和β1-integrin,这些结果表明IV型胶原粘附筛选的细胞中包含有干细胞。在此基础上,进一步选择生长状态良好的IV型胶原粘附后的第二代皮肤细胞作为克隆纯化的对象细胞,利用96孔板有限稀释法获得了形态较为原始,直径约为10μm的单细胞,经传代培养,该细胞能够形成典型的全克隆;而且,随传代次数增加,大多数细胞仍处于G1/G0期,保持着干细胞的慢周期性,有较高的克隆形成率,始终具有较大的增殖潜能和克隆遗传力;各代细胞冷冻复苏率与24小时贴壁率差异不显著（p<0.05）;大多数细胞的K15、K19、p63、CD34和β1-integrin等表面标志鉴定结果均呈阳性,分化标志K10和Involucrin检测结果呈阴性。在体外诱导实验中,这些纯化的干细胞能够被诱导生成神经元、神经胶质细胞、心肌细胞和成骨细胞,具有跨胚层分化潜能。以上结果充分证明了纯化的细胞就是绒山羊皮肤干细胞。此外,通过β₁-integrin和CD34的FISH初步定位了绒山羊皮肤干细胞。其中,β₁-integrin主要表达于绒山羊皮肤毛囊的外根鞘和毛乳头尖部,而CD34则主要表达于毛囊的外根鞘、毛乳头尖部和皮肤的基底层。更多还原

【Abstract】 In this study, we investigated the characteristics and the avenues of isolation, purification, identification and directional inducement of stem cells from adult cashmere goat skin.The skin sample of cashmere goat was incubated in 0.02% protease at 4℃overnight, and the epidermis and hair follicles were separated; then they were placed in a sterile tube containing 0.25% trypsin at 37℃for 30 min and the dissociated cells were adhered with collagen type IV to culture dishes for 10 min. 10min later, the unattached cells were removed and the rapidly adherent epidermal cells were cultured in stem cell growth medium. The cells showed a large nuclear: cytoplasmic ratio and small size, and can formed different shapes of clones. The cell cycle profiles for different passages suggested most of the rapidly adherent epidermal cells arrested at G1/G0 phase and they had a higher proliferative potential. Moreover, the isolated cells expressed k15, k19, p63, CD34 andβ1-Integrin. These results indicated that there were epidermal stem cells in the population of the rapidly adherent epidermal cells.Then, a single cell was isolated using 96-hole culture plate by diluting limitedly at the second passage of the rapidly adherent epidermal cells, and cultured for a single cell cloning and expanded in stem cell medium. The cells had the stem cell characteristics of immaturity, round, small size, few organelles, a high nuclear:cytoplasmic ratio, and they could form holoclone. The cells were slow cycle and most stayed at G1/G0 phase. The CFE of the cells was higher, and they possessed high proliferative capacity and strong clonogenicity in vitro. To evaluate the purified cell populations, further identification by immunocytochemistry and fluorescence in situ hybridization were performed, the results indicated they were positive for Keratin15, Keratin19, p63, CD34 andβ1-integrin, but negative for Keratin10 and Involucrin. This implied that the purifed cells can be skin stem cells. In vitro, the skin stem cellsof cashmere goats can differentiate into the cells with characteristics of cardiomyocytes, nerve, osteoblast, which suggested that the purifed cells had a big plasticity.Furthermore, the stem cells in cashmere goat skin were located primarily by FISH.β1-integrin were expressed positively in the outer root sheath of hair follicle and the papillae in cashmere goat skin, but CD34 were positive in the basal layer of epidermis, the outer root sheath of hair follicle and the papillae.更多还原