崂山奶山羊胎儿骨髓间充质干细胞(BMSCs)的分离培养及成神经诱导分化  

Isolation, Culture and Induced Neuronal Differentiation of Laoshan Dairy Goat Fetal Bone Marrow-derived Mesenchymal Stem Cells

作　　者：杨培培[1] 程明[1,2] 王学梅[1] 刘萌萌[1] 张倩[1] 戴正浩[1] 刘宗正[1,2] YANG Pei-pei CHENG Ming WANG Xue-mei LIU Meng-meng ZHANG Qian DAI Zheng-hao LIU Zong-zheng(Qingdao Municipal Institute for Animal Husbandry and Veterinary Medicine, Qingdao 266100, China Qingdao Aote Sheep Breeding Farm, Qingdao 266100, China)

机构地区：[1]青岛市畜牧兽医研究所,山东青岛266100 [2]青岛奥特种羊场,山东青岛266100

出　　处：《畜牧与饲料科学》2016年第9期99-103,共5页Animal Husbandry and Feed Science

基　　金：山东省现代农业产业技术体系羊产业创新团队青岛综合试验站(SDAIT-09-011-12);青岛市民生科技计划项目(14-2-3-45-nsh)

摘　　要：旨在建立崂山奶山羊胎儿骨髓间充质干细胞(BMSCs)体外分离培养方法,并研究其生物学特性和成神经分化的能力。取怀孕3个月的崂山奶山羊胎儿股骨,分离培养骨髓间充质干细胞,并进行传代培养,测定其细胞倍增时间,利用RT-PCR技术检测Oct4、Nanog、Sox2基因的表达;取P3 BMSCs分别向成神经细胞进行诱导分化,并从组织学水平和基因水平进行鉴定。结果表明,分离得到的胎儿骨髓间充质干细胞大小较为均匀,呈梭形的成纤维细胞样,可表达Oct4、Nanog、Sox2基因;传代接种后第4天进入指数生长期,第8天进入平台期,前10代BMSCs的平均倍增时间为29.7 h;P3 BMSCs成神经诱导后,尼氏体经甲苯胺蓝染色后可见紫蓝色,其特异性表达基因ENO2和GFAP表达呈阳性。获得的崂山奶山羊BMSCs具有成神经分化潜能。The aims of the present study were to establish the optimal in vitro method for isolating, purifying and proliferating of Laoshan dairy goat fetal bone marrow-derived mesenchymal stem cells （BMSCs） and to assess their biological characteristic and induced neuronal differentiation potentiality. The femur samples were collected from a three-month-old fetus of a female Laoshan dairy goat. The BMSCs were harvested by centrifuging and were subsequently purified and proliferated. The population doubling time （PDT） of the BMSCs were determined. RT-PCR assay was used to assess the presence of oct4, Nanog and Sox2 genes. Passage 3 cultures of the BMSCs were used to conduct induced neuronal differentiation experiment, and the induced products were histologically and genetically identified. The results showed that the morphology of the isolated cells were uniform and was fibroblasts-liking spindle and the presence of oct4, Nanog and Sox2 genes were detected. After sub-cultured, the growth cells entered exponential growth phase on the 4th day and subsequently entered platform growth phase on the 8th day. The PDT of the first 10 passage cultures was 29.7 h. After induced neuronal differentiation, the purple-blue Nissl body was observed by toluidine blue staining method in passage 3 cultures. The specific expression of ENO2 and GFAP genes were positive in the induced cells. The results demonstrated that the BMSCs of Laoshan dairy goat is potential to be differentiated to neurons.

关 键 词：崂山奶山羊 胎儿 骨髓间充质干细胞 诱导分化 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] S827.3[医药卫生—基础医学]