奶山羊成肌细胞体外分离培养及鉴定  

Isolation, Culture and Identification of Dairy Goat(Capra hircus)Myoblast In vitro

作　　者：吕明 罗军[1] 石恒波[2] 田慧彬 王平[1] LV Ming;LUO Jun;SHI Heng-Bo;TIAN Hui-Bin;WANG Ping(College of Animal Science and Technology,Northwest A＆F University,Yangling 712100,China;College of Life Sciences,Zhejiang Sci-Tech University,Hangzhou 318100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]浙江理工大学生命科学学院,杭州318100

出　　处：《农业生物技术学报》2018年第11期1996-2002,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31501983);国家重点研发计划(No.2017YFD0502005);陕西省重点研发计划项目(No.2018ZDXM-NY-033)

摘　　要：成肌细胞(myoblast)具有自我更新和多向分化的能力,对骨骼肌的再生和修复起着重要作用。为了建立奶山羊(Capra hircus)肌细胞的体外分离纯化、培养和鉴定的方法,了解其增殖和成肌特性,为奶山羊肌肉发育调控机理研究提供实验材料,本研究采集西农萨能奶山羊羔羊骨骼肌组织,采用I型胶原酶和胰酶两步酶消化法分离细胞,结合差速贴壁纯化得到成肌细胞,利用qRT-PCR和免疫荧光染色法对分离纯化后的成肌细胞进行进一步鉴定。细胞形态学观察结果显示,分离纯化后的成肌细胞呈梭形,生长状态良好;qRT-PCR和免疫荧光染色结果显示,生肌调节因子5(myogenic regulatory factor5, Myf5)和成肌决定因子(myogenic differentiation factor, MyoD)、结蛋白(desmin)呈阳性表达,纯化后的成肌细胞纯度大于95%;诱导分化后的成肌细胞能够相互融合形成肌管,成肌特异性标志蛋白肌球蛋白重链(myosin heavy chain, MYHC)呈阳性表达。本研究建立了奶山羊成肌细胞体外分离培养的方法,并成功获得了高纯度的具有增殖和成肌特性的成肌细胞,可用于奶山羊肌肉发育调控研究。Myoblast is capable of self-renewal and polymorphism, and plays an important role in the regeneration and repair of skeletal muscle. In order to establish a method for isolation and purification, culture and identification of dairy goat （Capra hircus） myoblasts in vitro, and to understand the identification of myoblast features of proliferation and myogenesis, and to provide experimental materials for the regulation mechanism of muscle development in dairy goats, the samples were taken from skeletal muscle of Xinong Saanen dairy goat kids. Cells were disassociated with collagenase I and trypsin following with purification of cell using differential adhesion approach. qRT-PCR and immunofluorescence staining were conducted to further identify purified cells. Cell morphological results showed that purified cells were in spindle shape and maintained a stable status of growth. Myogenic regulatory factors, myogenic differentiation factor （MyoD） and myogenic regulatory factor 5 （Myf5）, and desmin were positively expressed in purified cells （percentage of purified cell 〉 95%） as observed from the results of qRT-PCR and immunofluorescence staining. Purified myoblasts were able to fuse into myotubes after induced differentiation. Myosin heavy chain （MYHC）, a specific marker of differentiated myotubes, was positively expressed. This study establish a method of isolation and culture of dairy goat myoblasts in vitro. Myoblast line with high purity is successfully obtained, which is able to be used as an experimental material in studies of regulation mechanism of muscle development in dairy goats.

关 键 词：奶山羊 成肌细胞 分离 鉴定 

分 类 号：S827[农业科学—畜牧学]