马脐带间充质干细胞的分离培养与生物学特性研究  

Isolation,culture and biological characterization of horse umbilical cord-derived mesenchymal stem cells

作　　者：江文康 黄国健 雷彬 王彩莹 罗惠娜 赵明明 樊全宝 阮慧敏 陈胜锋 王丙云 JIANGWen-kang;HUANG Guo-jian;LEI Bin;WANG Cai-ying;LUO Hui-na;ZHAO Ming-ming;FAN Quan-bao;RUAN Hui-min;CHEN Sheng-feng;WANG Bing-yun(College of Life Science and Engineering,Foshan University of Science and Technology,Foshan,Guangdong 528231,China;Huangcun Sports Training Center of Guangdong,Guangzhou 510663,China;Guangdong VetCell Bio-tech Co.,Ltd.,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 [2]广东省黄村体育训练中心,广州510663 [3]广东维赛生物科技有限公司,广东佛山528231

出　　处：《南方农业学报》2021年第7期1998-2006,共9页Journal of Southern Agriculture

基　　金：广东省自然科学基金项目(2017A030313171,2018A030313892);广东省普通高校动物干细胞工程技术研究中心项目(2021GCZX006)。

摘　　要：【目的】建立马脐带间充质干细胞(UC-MSCs)体外分离培养体系,研究其增殖能力、分化能力和生物学特性,为推广UC-MSCs在赛马运动损伤治疗方面的应用提供理论依据。【方法】采用I型胶原酶消化法从脐带组织分离马UC-MSCs,通过绘制生长曲线及计算群体倍增时间检测其体外增殖能力,结合流式细胞仪检测和RT-PCR扩增鉴定表面标志物(CD29、CD44、CD45、CD73、CD90和CD105),并通过体外成脂成骨诱导分化检测其分化潜能。【结果】分离获得的原代马UC-MSCs为折光性强的圆形悬浮细胞,培养10 d后细胞融合达90%,且传代后细胞增殖速度明显提高,传代至P3代,细胞呈旋涡状生长,形态为均一的长梭形,生长趋势符合Logistic生长曲线规律,呈典型的S形。流式细胞仪检测结果显示,马UC-MSCs高表达CD29、CD44和CD90,表达率分别为98.45%、97.08%和96.56%,呈强阳性,但不表达CD45;RT-PCR扩增结果表明,马UC-MSCs表达CD29、CD44、CD73、CD90和CD105等MSCs表面标志物基因,但不表达CD45基因。以胰岛素、IBMX、罗格列酮和地塞米松为主要试剂进行马UC-MSCs体外成脂诱导,诱导第10 d发现细胞内有小脂滴形成,至诱导第18 d有大量脂滴形成;选用抗坏血酸、β-甘油磷酸钠和地塞米松为主要试剂进行体外成骨诱导,至诱导第7 d可观察到钙结节形成。【结论】采用I型胶原酶消化法分离获得的马UC-MSCs纯度高,且具有良好的体外增殖能力和多向分化潜能;掌握好马UC-MSCs的有效冻存方式,可为治疗赛马运动损伤提供优质的种子细胞。【Objective】The in vitro isolation and culture system of horse umbilical cord-derived mesenchymal stem cells(UC-MSCs)has been established to study its proliferative ability,differentiation ability and biological characteris-tics,and provide a theoretical basis for promoting the application of UC-MSCs in the treatment of horse racing sports in-jury.【Method】Horse UC-MSCs were obtained by using type I collagenase digestion,and cell proliferation in vitro was determined by drawing growth curve and calculating population doubling time.Cell surface markers(CD29,CD44,CD45,CD73,CD90 and CD105)were identified by flow cytometry and RT-PCR amplification.The differentiation potential was detected by inducing adipose formation and osteogenesis differentiation in vitro.【Result】The results revealed the isolated primary horse UC-MSCs was a round suspension cell with strong refraction.After 10 d of culture,the cell fusion reached 90%,and the cell proliferation rate increased after passage.After passage to the P3 generation,the cells grew in a vortex shape with a uniform long fusiform shape,and the growth trend was in accordance with the law of Logistic growth curve,showing a typical S shape.The results of flow cytometry showed that horse UC-MSCs highly expressed CD29,CD44 and CD90,and the expression rates were 98.45%,97.08%and 96.56%,respectively,which were highly positive.But CD45 was not expressed.The results of RT-PCR showed that horse UC-MSCs expressed MSCs surface marker genes such as CD29,CD44,CD73,CD90 and CD105,but did not express CD45 gene.Adipogenesis of horse UC-MSCs was induced in vitro with insulin,IBMX,rosiglitazone and dexamethasone as main reagents.Small lipid droplets were found on the 10th day of induction,and a large number of lipid droplets formed on the 18th d of induction.Ascorbic acid,sodiumβ-gly cerophosphate and dexamethasone were used as the main reagents for osteogenic induction in vitro.Calcium nodule forma-tion was observed on the 7th d of induction.【Conclusion】The results show that th

关 键 词：马 脐带 间充质干细胞 生物学特性 I型胶原酶消化法 

分 类 号：S821.91[农业科学—畜牧学]