昆明白小鼠胚胎生殖细胞的分离与培养  

Isolation and in Vitro Culture of Embryonic Germ Cells (EG) from Kunming White Mice

作　　者：赵向远[1] 李洋 刘延玲 董焕声[1] 王红军[1] 董晓[2] ZHAO Xiangyuan;LI Yang;LIU Yanling;DONG Huansheng;WANG Hongjun;DONG Xiao(College of Animal Science,Qingdao Agricultural University,Qingdao 266109,China;College of Life Science,Qingdao Agricultural University;Dongcheng Street Health Center,Linqu County,Weifang 262600,China)

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]青岛农业大学生命科学学院 [3]临朐县东城街道卫生院,山东潍坊262600

出　　处：《青岛农业大学学报（自然科学版）》2019年第2期85-90,102,共7页Journal of Qingdao Agricultural University(Natural Science)

基　　金：青岛农业大学高层次人才科研基金(6631117017);山东省自然科学基金2017年面上项目(ZR201702210030)

摘　　要：胚胎生殖细胞(embryonic germ cells,EG)是来源于原始生殖细胞(primordial germ cells,PGC)的多潜能干细胞。本研究旨在优化昆明白小鼠EG细胞的培养条件。以昆明白小鼠10.5dpc胚胎PGC为材料,以丝裂霉素C处理的小鼠成纤维细胞(mouse embryonic germcells,MEF)为饲养层,培养小鼠EG细胞,以对影响小鼠EG细胞传代的因素进行探讨。用ELISA试剂盒定量检测MEF自身分泌生长因子SCF、bFGF、LIF的浓度;以培养液是否添加生长因子,观察PGC/EG的培养效果;比较了机械分割法、胰酶消化法和胶原酶Ⅳ消化法对EG传代培养的影响;对获得的EG进行多能性鉴定。结果表明,培养3d的饲养层上清液中含75.7ng/LSCF、125.7ng/LbFGF、196.6ng/LLIF,培养5d的饲养层上清液中含76.2ng/LSCF、136.2ng/LbFGF、214.2ng/LLIF;培养液添加生长因子显著提高了EG的克隆数、传代数(P<0.05);机械分割法与酶消化法对EG传代差异显著(P<0.05),而两种酶法对EG传代无显著影响(P>0.05);所分离的EG细胞显示AKP染色强阳性;EG体外培养时会自分化为神经样细胞、上皮样细胞;EG细胞呈Oct4、SSEA-1的免疫荧光检测阳性。结果提示,饲养层自身能分泌少量生长因子,但培养液中添加生长因子可显著改善昆明白小鼠EG培养效果,酶消化法更适合于昆明白小鼠EG的传代培养。Embryonic germ cells (EG) are pluripotent stem cells derived from primordial germ cells (PGC). The goal of this study was to optimize in vitro culture conditions for EG from Kunming White mice. Mitomycin C treated mouse embryonic fibroblasts ( MEF ) were used as feeder cells. Firstly,the amounts of SCF,bFGF,LIF secreted by MEF were measured by ELISA;Then,the proliferation of PGC/EG in presence of growth factors ( SCF,bFGF,LIF ) or not were measured;EG were passaged with mechanical segmentation or enzymatic dissociation to determine the most suitable passage method;Finally,the pluripotency of EG under different culture conditions were identified. The results showed,after 3 days in vitro culture,the supernatant of MEF contained 75.7 ng/L SCF ,125.7 ng/L bFGF ,196.6 ng/L LIF ,respectively;While at day 5,the supernatant of MEF contained 76.2 ng/L SCF ,136.2 ng/L bFGF ,214.2 ng/L LIF;Addition of growth factors significantly increased EG colony numbers and passage numbers ( P <0.05);Mechanical segmentation and enzymatic dissociation showed significant different effects on EG passage ( P <0.05),while different enzymes (0.25% trypsin+0.04% EDTA or 0.1% collagenase IV) had no difference on EG passage ( P >0.05);The cultured EG showed strong AKP activity,spontaneously differentiated into neuroblast-like and epithelial-like cells,and positive staining for Oct4 and SSEA-1 immunofluorescence identification. In conclusion,the feeders can secrete little growth factors,and the culture effects of EG is much better when adding growth factors to the culture medium. The enzymatic dissociation is more suitable for Kunming White mice EG passage.

关 键 词：胚胎生殖细胞 饲养层 生长因子 传代方法 分化 

分 类 号：S813.1[农业科学—畜牧学]