| 马脐带间充质干细胞的分离培养与生物学特性研究 |
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| 引用本文： | 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.马脐带间充质干细胞的分离培养与生物学特性研究[J].南方农业学报,2021,52(7):1998-2006. |
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| 作者姓名： | 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 |
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| 作者单位： | 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 |
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| 基金项目： | 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） |
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| 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。
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| 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 |
| 收稿时间： | 2020-12-20 |
Isolation,culture and biological characterization of horse umbilical cord-derived mesenchymal stem cells |
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| JIANG Wen-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.Isolation,culture and biological characterization of horse umbilical cord-derived mesenchymal stem cells[J].Journal of Southern Agriculture,2021,52(7):1998-2006. |
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| Authors: | JIANG Wen-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 |
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| Affiliation: | 1 College of Life Science and Engineering, Foshan University of Science and Technology, Foshan, Guangdong 528231, China;2 Huangcun Sports Training Center of Guangdong, Guangzhou 510663, China;3 Guangdong VetCell Bio-tech Co., Ltd., Foshan, Guangdong 528231, China |
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| Abstract: | 【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 characteristics, and provide a theoretical basis for promoting the application of UC-MSCs in the treatment of horse racing sports injury.【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, sodium β-glycerophosphate and dexamethasone were used as the main reagents for osteogenic induction in vitro. Calcium nodule formation was observed on the 7th d of induction.【Conclusion】 The results show that the purity of the horse UC-MSCs obtained by I-type collagen enzyme digestion is high and has good in vitro proliferation and multi-directional potential;master the effective freezing mode of horses UC-MSCS, providing quality seed cells in the treatment of horse. |
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