奶牛脂肪来源间充质干细胞的分离培养及其生物学特性研究  

Isolation,culture and biological characteristics of dairy cow adipose-derived mesenchymal stem cells

作　　者：罗惠娜 樊全宝 江文康 赵明明 王静静 罗冬章 王丙云 LUO Hui-na;FAN Quan-bao;JIANGWen-kang;ZHAO Ming-ming;WANG Jing-jing;LUO Dong-zhang;WANG Bing-yun(College of Life Science and Engineering,Foshan University,Foshan,Guangdong 528231,China)

机构地区：[1]佛山科学技术学院生命科学与工程学院,广东佛山528231

出　　处：《南方农业学报》2020年第6期1462-1469,共8页Journal of Southern Agriculture

基　　金：广东省自然科学基金项目(2020A1515011110,2018A030313892,2017A030313171)。

摘　　要：【目的】建立奶牛脂肪间充质干细胞(AD-MSCs)体外分离培养体系,并进行增殖能力检测、分化能力鉴定及生物学特性研究,为推广AD-MSCs在畜牧生产及其疾病治疗等方面的应用提供理论依据。【方法】通过I型胶原酶消化法分离奶牛AD-MSCs并进行代传培养,绘制P3、P6和P9代奶牛AD-MSCs的生长曲线及测定其群体倍增时间,分别采用流式细胞术及RT-PCR检测细胞表面标志物;使用干细胞成骨/成脂诱导分化完全培养基进行奶牛AD-MSCs成骨成脂诱导分化,经茜素红和油红O染色后鉴定其成骨成脂分化能力;并检测冻存奶牛AD-MSCs复苏后的存活率。【结果】分离及传代培养获得的奶牛AD-MSCs在体外培养条件下呈长梭形,折光性强,生长状态良好,其生长曲线呈典型的S形,符合Logistic生长规律。奶牛AD-MSCs高表达MSCs表面标志物CD44、CD73、CD90和CD105,但不表达白细胞表面标志物CD45;分别使用干细胞成骨/成脂诱导分化完全培养基诱导的奶牛AD-MSCs经茜素红和油红O染色后,显微镜下可观察到大量的钙结节和红色脂肪滴。冻存6个月后进行细胞复苏,奶牛AD-MSCs的存活率高达96%;复苏奶牛AD-MSCs培养4 h内贴壁,呈典型的长梭形,生长状态良好,培养72 h细胞融合达80%~90%。【结论】通过I型胶原酶消化法从奶牛腹部脂肪组织分离获得的奶牛AD-MSCs具有良好的体外增殖能力及多向分化潜能,为畜牧生产研究及奶牛疾病治疗提供了一种良好的种子细胞来源。【Objective】This study aimed to establish in vitro culture system for dairy cow adipose-derived mesenchymal stem cells(AD-MSCs)and conduct proliferation ability detection,differentiation ability identification and the biological characteristics of these cells,provide a theoretical basis for promoting the application of AD-MSCs in animal husbandry production and disease treatment.【Method】Collagenase type I digestion method was applied to separate dairy cow ADMSCs and then sub-cultured in vitro,and then plotted the growth curves,determined its population doubling time of P3,P6 and P9 generation of AD-MSCs.Membrane markers of the cells were identified by flow cytometric analysis and RTPCR.The ability of osteogenic/adipogenic differentiation was detected by alizarin red stainingand oil red O,then livability of AD-MSCs in cryopreserved cow milk after resuscitation.【Result】The results revealed that the cultured dairy cow ADMSCs presented typical long spindle shape,strong refractivity and fine growth status.The growth curve showed a typical S shape,which was in line with the law of Logistic growth curve.The MSCs surface markers CD44,CD73,CD90 and CD105 were highly expressed,while white blood cell surface marker CD45 was rarely detected.After being stained with alizarin red and oil red O using stem cell osteogenic/adipogenic differentiation-inducing complete media,a large number of calcium nodules and red fat droplets were observed under the microscope.After 6 months of cryopreservation,the cells were recovered and the cell survival rate was as high as 96%.The recovered AD-MSCs adhered to the wall after 4 h of cul ture and showed a typical long spindle shape with good growth status.The AD-MSCs were confluent by 80%-90%after 72 h of culture.【Conclusion】The AD-MSCs of cows isolated from the abdomen adipose tissue of cows by type I collagenase digestion method have good in vitro proliferation ability and multi-directional differentiation potential,which provides a good seed cell source for animal husbandry pr

关 键 词：奶牛 脂肪 间充质干细胞(MSCs) 生物学特性 体外增殖能力 分化潜能 

分 类 号：S823.89[农业科学—畜牧学]