悬滴培养法促进鸡胚胎干细胞形成类胚体  

Embryoid Body Formation from Chicken Embryonic Stem Cells through Suspension Culture

作　　者：张蕾[1,2] 王宵燕[1] 左其生[1] 路镇宇 王飞[1] 纪艳芹 王颖洁[1] 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院江苏省动物遗传繁育与分子设计重点实验室,扬州225009 [2]江苏农牧科技职业学院,泰州225300

出　　处：《畜牧兽医学报》2015年第8期1333-1340,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31272429;31472087);江苏省研究生科研创新基金(CXZZ13_0909)

摘　　要：本研究旨在通过悬浮培养法培养鸡胚胎干细胞(Embryonic stem cells,ESCs),摸索鸡胚胎干细胞形成类胚体(Embryoid body,EB)的最佳方案,以期提高鸡胚胎干细胞体外诱导效率。将鸡ESCs培养传代至第3代,采用1×104、3×104和6×104 mL-13个细胞浓度,使用悬滴培养后,观察类胚体的形成效率。对悬滴培养形成的类胚体进行形态学观察,qRT-PCR检测干细胞标记基因的表达,免疫细胞化学检测相关蛋白的表达,并进行类胚体自分化检测和核型分析。3×104 mL-1细胞浓度生成的类胚体数量最高,单个视野下可观察到约267个类胚体,且形成的类胚体大小均一,呈圆球状。qRT-PCR检测结果表明,在类胚体形成48h内,干细胞标记基因Nanog、Sox2、Oct4和C-kit仍维持表达。免疫细胞化学检测显示,该方法形成的类胚体表面抗原检测Nanog和SSEA-1呈阳性。自分化检测三胚层分化标记基因SOX17、SMA和TUJ-1呈阳性。核型分析显示,悬滴培养形成的类胚体具有并保持正常的核型。鸡ESCs悬滴培养形成类胚体的适宜细胞浓度为3×104 mL-1,且形成的类胚体可分化成3个胚层,用于体外定向诱导过程。本研究建立了鸡ESCs体外悬滴培养形成类胚体的培养体系,为后期信号通路的体外验证提供试验基础。This experiment was conducted to explore the best suspension culture system for embryoid body(EB)formation from chicken embryonic stem cells(ESCs),and to improve the induction efficiency of chicken ESCs in vitro.Pure clones from the third generation of chicken ESCs was collected,re-suspensed the cells into 3cell concentrations:1×104,3×104 and 6×104 mL-1 to make suspension culture.Morphology changes of EB were observed,qRT-PCR and immunofluorescence methods were used to detect the marker genes' s expression,self-differentiation and karyotype analysis were made to make full test of EB.The results showed that,the amount of EB with3×104 mL-1 cell concentration was higher than the other groups,and the number of EB reached267 in one single view with the same spherical shape.The stem cell marker genes Nanog,Sox2,Oct4 and C-kit remained expression with 48 hof EB formation,and stem cell surface specific anti-gen(Nanogand SSEA-1)detection was positive.After self-differentiation of EB,3embryonic germ layers specific antigen(SOX17,SMAand TUJ-1)detection all showed positive.Karyotype analysis showed that the formatted EB maintained normal karyotype.The results indicated that the suitable concentration for chicken ESCs form the EB through suspension culture was 3×104mL-1,and the formatted EB had viability to provide experimental foundation for chicken ESCs induction i n vitro.

关 键 词：类胚体 悬滴培养 鸡 胚胎干细胞 

分 类 号：S831[农业科学—畜牧学]