阿尔巴斯绒山羊胎儿皮肤干细胞的分离培养及鉴定  

Isolation and Identifi cation of Fetal Skin Stem Cells from Arbas Cashmere Goat

作　　者：乃门塔娜 梁红宇[1] 仓明[1] 郭旭东[1] 刘东军[1] 旭日干[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010070

出　　处：《中国细胞生物学学报》2016年第2期143-149,共7页Chinese Journal of Cell Biology

基　　金：国家科技重大专项(批准号:2014ZX08008-002)资助的课题~~

摘　　要：该研究旨在体外分离培养和鉴定阿尔巴斯绒山羊胎儿皮肤干细胞,为绒毛生长相关研究提供实验材料。以阿尔巴斯绒山羊胎儿皮肤为样本,酶消化和IV型胶原黏附法获得较纯的皮肤干细胞。通过形态学观察、生长曲线、细胞免疫组化、实时定量PCR和体外分化等方法探讨皮肤干细胞体外培养方法和生物学特性。结果发现,皮肤干细胞形态较小,铺路状生长,折光度高且黏附能力强,在体外培养至20代未出现明显细胞形态变化。免疫组化染色显示,胎儿皮肤干细胞阳性表达Krt15、CD34和Itgβ1。Krt15、CD34和Itgβ1 m RNA相对水平分别为绒山羊角质细胞的5.56、24.37和7.22倍(P<0.01)。体外成骨诱导Von Kossa染色呈阳性;成软骨诱导阿尔新蓝染色呈阳性;成肌诱导后,Hoechst 33342染色观察到细胞融合的发生,Myo G免疫组化染色呈阳性。结果表明,成功建立了阿尔巴斯绒山羊胎儿皮肤干细胞系。In this study, fetal skin stem cells from Arbas Cashmere goat were isolated and cultured in vitro, in order to providing experimental materials for further research on hair follicle growth. Highly purified skin stem cells were isolated using enzyme digestion and adhesion to type IV collagen. The biological characteristics of skin stem cells were identified by morphological observation, growth curve, markers assay and differentiation in vitro. The results showed that the cells were in small cell size with typical cobblestone-like morphology, good adhesion capacity and high refractivity, and no significant morphological changes were appeared after 20 passages. Immunocytochemistry results showed the cells were positively expressed Krt15, CD34 and Itgβ1. The relative m RNA levels of Krt15, CD34 and Itgβ1 were 5.56, 24.37 and 7.22 times of their levels in Cashmere goat keratinocytes, respectively（P〈0.01）. After osteogenic and cartilage induction, the cells were positively stained by Von Kossa and alcian blue, respectively. The cells positively expressed Myo G after myogenic differentiation and cell fusion was observed by Hoechst 33342 staining. These results suggested that Arbas Cashmere goat fetal skin stem cell line was successfully established.

关 键 词：阿尔巴斯绒山羊 皮肤干细胞 细胞免疫组化 实时定量PCR 体外分化 

分 类 号：S827[农业科学—畜牧学]