中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (13): 2020-2026.doi: 10.3969/j.issn.2095-4344.0496

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

无血清和有血清培养人脐带间充质干细胞的对比

张学娟1，2，刘高米洋2，刘菊芬2，宋乙甲1，2，林庆铿1，2，白盈盈1，2，潘兴华2   

  1. 1昆明医科大学解放军昆明总医院临床学院，云南省昆明市 650032；2解放军昆明总医院细胞生物治疗中心，干细胞与免疫细胞生物医药技术国家地方联合工程实验室，云南省细胞治疗技术转化医学重点实验室，云南省昆明市 650032
  • 修回日期:2018-02-02 出版日期:2018-05-08 发布日期:2018-05-08
  • 通讯作者: 潘兴华，博士后，主任医师，教授，解放军昆明总医院细胞生物治疗中心，干细胞与免疫细胞生物医药技术国家地方联合工程实验室，云南省细胞治疗技术转化医学重点实验室，云南省昆明市 650032
  • 作者简介:张学娟，女，1993年生，云南省大理州人，白族，昆明医科大学在读硕士，主要从事人脐带间充质干细胞与衰老相关研究。 并列第一作者：刘高米洋，女，1988年生，云南省宣威市人，汉族，2014年海军军医大学(原第二军医大学)毕业，硕士，医师，主要从事脐带间充质干细胞的临床转化研究。
  • 基金资助:

    云南省细胞治疗技术转化医学重点实验室(2015DG034)；全军实验动物专项(SYDW(2016)004)

A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells

Zhang Xue-juan1, 2, Liu Gao-mi-yang2, Liu Ju-fen2, Song Yi-jia1, 2, Lin Qing-keng1, 2, Bai Ying-ying1, 2, Pan Xing-hua2   

  1. 1Kunming General Hospital Clinical College of Kunming Medical University, Kunming 650032, Yunnan Province, China; 2Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China
  • Revised:2018-02-02 Online:2018-05-08 Published:2018-05-08
  • Contact: Pan Xing-hua, M.D., Chief physician, Professor, Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China
  • About author:Zhang Xue-juan, Master candidate, Kunming General Hospital Clinical College of Kunming Medical University, Kunming 650032, Yunnan Province, China; Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China. Liu Gao-mi-yang, Master, Physician, Cell Biological Therapy Center of Kunming General Hospital of PLA, Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan Province, the Stem Cell Therapy Key Laboratory of Yunnan Province, Kunming 650032, Yunnan Province, China. Zhang Xue-juan and Liu Gao-mi-yang contributed equally to this work.
  • Supported by:

    the Project of the Stem Cell Therapy Key Laboratory of Yunnan Province, No. 2015DG034; the Experimental Animal Specific Fund of PLA, No. SYDW(2016)004

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摘要：

文章快速阅读：

文题释义：
胎牛血清：含有细胞生长所需的基本营养成分和丰富的生物活性因子(如生长因子、激素、多肽类物质等)，在细胞的代谢、增殖与分化中发挥着重要的调节作用，用于临床规模生产间充质干细胞的大多数分离和扩增方案均使用补充有体积分数为10%胎牛血清的培养基。
血清替代物：不含异种血清，但包含细胞生长所需的营养素、细胞因子，可避免免疫排斥、病毒传播等问题，用血清替代物培养的细胞上清可直接用来提取外泌体，排除血清的影响。

 

摘要
背景：研究表明有血清培养体系存在若干风险和问题，比如免疫排斥、批次差异、病毒风险等，另外随着外泌体的发现和应用，无血清培养体系变得越来越重要。
目的：系统比较人脐带间充质干细胞无血清培养体系与传统有血清培养体系的异同，为人脐带间充质干细胞的临床转化奠定基础和提供实验数据。
方法：无菌条件下采集健康剖腹产足月儿脐带，组织块贴壁法培养人脐带间充质干细胞，从第1代开始有血清培养组用体积分数为10%胎牛血清培养，血清替代物组用体积分数为15%血清替代物培养。倒置显微镜观察其形态变化，流式细胞仪检测其表面标记物，CCK-8检测其增殖能力，诱导分化实验检测其多向分化潜能， Western Blot检测干性标志物oct4、nanog、sox2的蛋白水平。
结果与结论：①倒置显微镜下观察可见血清替代物组人脐带间充质干细胞呈现较均一的漩涡状生长，胎牛血清组人脐带间充质干细胞随着细胞代数的增加逐渐出现细胞分化或者老化现象；②两种培养法培养的人脐带间充质干细胞均表达CD73，CD90和CD105，低表达CD34和CD45，二者无明显差异；③增殖能力上胎牛血清组优于血清替代物组；④两种培养法培养的人脐带间充质干细胞均具有成脂、成骨、成软骨诱导分化能力，二者无明显差异；⑤血清替代物组与胎牛血清组oct4、nanog的表达水平无显著差异，血清替代物组sox2表达水平高于胎牛血清组(P < 0.05)；⑥血清替代物培养的人脐带间充质干细胞符合间充质干细胞国际标准，血清替代物能够替代胎牛血清成为培养人脐带间充质干细胞的优选方法。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0003-2572-725X(张学娟)

关键词: 脐带间充质干细胞, 胎牛血清, 血清替代物, 外泌体, 干细胞

Abstract:

BACKGROUND: Studies have shown increasing risks and problems in the serum culture system, such as immune rejection, batch differences and virus risk. In addition, with the discovery and application of exosomes, the serum-free culture system is becoming an increasing concern.
OBJECTIVE: To compare the similarities and differences between the serum-free culture system and the traditional serum culture system, which lays the foundation for the clinical transformation of human umbilical cord mesenchymal stem cells (hUCMSCs) and provides experimental data.
METHODS: Umbilical cord was collected from term infants of cesarean section under aseptic condition, and hUCMSCs were isolated and cultured by explant tissue technique. hUCMSCs was cultured with 10% fetal bovine serum (FBS) and 15% serum substitutes (AGS) from the original generation. Then an inverted microscope was used to observe cell morphological changes. Flow cytometry was used to detect cell surface markers. Cell counting kit-8 was used to detect cell proliferation. Induced differentiation experiment was used to detect cell differentiation potential. Western Blot was used to detect the protein levels of oct4, nanog and sox2.
RESULTS AND CONCLUSION: Under the inverted microscope, hUCMSCs cultured with AGS showed more uniform vortex-like growth, and those cultured with FBS gradually appeared with cell differentiation or aging with the increase of cell generations. hUCMSCs cultured by both methods expressed CD73,CD90 and CD105 but lowly expressed CD34 and CD45, and there was no significant difference between the two culture methods. FBS method was superior to AGS method in proliferation ability. Results from the induced differentiation experiments showed that hUCMSCs cultured by both methods had adipogenic, osteogenic and chondrogenic abilities, and there was no significant difference between the two culture methods. hUCMSC cultured by both methods expressed oct4 and nanog but showed no significant difference in level, while the expression of sox2 was significantly higher in the hUCMSCs cultured by AGS than by FBS (P < 0.05). To conclude, the hUCMSCs cultured with AGS are in accordance with the international standards of mesenchymal stem cells. The AGS method as an alternative to the FBS method can become a preferred method for hUCMSCs culture.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Serum, Tissue Engineering

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