奶牛脂肪间充质干细胞体外分离培养与鉴定  

Isolation,culture and identification of bovine adipose-derived mesenchymal stem cells in vitro

作　　者：鲁文赓[1] 徐郑美 刘庆 于楠楠[2] 于佳斌 李维龙 毛莹莹 曹立明 杜珍珍 司淋清 付世新[1] 韩英浩[2] LU Wen-geng;XU Zheng-mei;LIU Qing;YU Nan-nan;YU Jia-bin;LI Wei-long;MAO Ying-ying;CAO Li-ming;DU Zhen-zhen;SI Lin-qing;FU Shi-xin;HAN Ying-hao(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang 163319,China;College of Life Science&Biotechnology Heilongjiang Bayi Agricultural University,Daqing,Heilongjiang 163319,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319

出　　处：《中国兽医学报》2020年第5期1042-1046,1058,共6页Chinese Journal of Veterinary Science

基　　金：研究生创新科研资助项目(YJSCX2018-Y19);黑龙江省农垦总局重点科研资助项目(HKKY190302);黑龙江八一农垦大学“三纵三横”科研人才支持计划—基础培育计划资助项目(ZRCPY201808)。

摘　　要：探讨奶牛脂肪来源的间充质干细胞(adipose-derived mesenchymal stem cells,AD-MSCs)体外分离培养方法及其生物学特性。采集不同部位奶牛脂肪,选用0.25%胰蛋白酶消化,并进行贴壁培养,获取原代奶牛AD-MSCs,通过传代培养进一步纯化扩增。显微镜观察细胞形态及生长特征,并采用MTT法绘制2种AD-MSCs生长曲线,利用免疫荧光化学染色法检测其表面标志物,对该细胞进行了向成骨及成脂诱导分化培养试验,采用油红O和钙盐染色法对其分化能力进行鉴定。比较研究了不同部位脂肪的形态,2种不同部位的AD-MSCs均呈长梭形、纺锤形或多角形贴壁生长,且2个部位来源的AD-MSCs的生长曲线无明显差异(P>0.05)。分离培养的AD-MSCs的表面标志物CD44和CD73呈阳性表达,而CD34和CD45呈阴性表达。在诱导分化培养试验中呈现出较强的成脂和成骨分化能力。本试验选取不同部位的脂肪组织,成功的分离并鉴定了奶牛AD-MSCs,为纯化鉴定奶牛AD-MSCs提供了依据。To explore the isolation,culture and biological characteristics of bovine adipose-derived mesenchymal stem cells(AD-MSCs) in vitro.Adipose tissue from two different parts was collected in the cows,adipose tissue was digested with 0.25% trypsin,and adherent culture was performed to obtain AD-MSCs,and then the AD-MSCs were further purified and amplified through subculture.The morphology and growth characteristics of the AD-MSCs were observed by microscope,and the growth curves of two kinds of AD-MSCs were drawn by MTT assay.The surface markers of AD-MSCs were detected by immunofluorescence staining.The AD-MSCs were cultured for osteogenesis and adipogenic induction differentiation,and then the differentiation ability of AD-MSCs was evaluated by oil red O and calcium deposition staining.The primary and passaged AD-MSCs from the two different parts were the shape of long shuttle,spindle or polygonal-shaped and adherent growth,and the growth curves of AD-MSCs from the two parts had no significant difference(P>0.05).The surface markers CD44 and CD73 of cultured AD-MSCs were positively expressed,while CD34 and CD45 were negatively expressed.The ability of adipogenic and osteogenic differentiation was strong in the induction and differentiation culture experiment.In this experiment,bovine AD-MSCs are successfully isolated and identified,which provided data support for the purification and identification of AD-MSCs in dairy cows.

关 键 词：奶牛 间充质干细胞 体外分离培养 体外诱导分化 

分 类 号：Q813.1[生物学—生物工程]