中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (24): 3814-3820.doi: 10.12307/2022.559

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

提取人骨髓间充质干细胞的新方法与传统方法比较

李石旦1，邢  伟2，谢晓瑜1，李又彬1，王绍川1，费  军3   

  1. 陆军军医大学陆军特色医学中心，1骨科，2野战外科研究所，3急诊科，重庆市  400002
  • 收稿日期:2021-07-21 接受日期:2021-09-06 出版日期:2022-08-28 发布日期:2022-01-22
  • 通讯作者: 费军，博士后，副主任医师，陆军军医大学陆军特色医学中心急诊科，重庆市 400002
  • 作者简介:李石旦，男，1993年生，湖南省岳阳市人，汉族，陆军军医大学在读硕士，主治医师，主要从事骨感染与骨康复医学研究。
  • 基金资助:
    陆军军医大学科技创新能力提升专项资助(2019XLC2023)，项目负责人：费军；重庆市技术创新与应用发展专项面上项目(cstc2019jscx-msxmX0210)，项目参与人：李又彬，王绍川

A new method for extracting human bone marrow mesenchymal stem cells and the comparison with traditional methods

Li Shidan1, Xing Wei2, Xie Xiaoyu1, Li Youbin1, Wang Shaochuan1, Fei Jun3   

  1. 1Department of Orthopedics, 2Research Institute of Battle Surgery, 3Department of Emergency, Army Specialty Medical Center, Army Medical University, Chongqing 400002, China
  • Received:2021-07-21 Accepted:2021-09-06 Online:2022-08-28 Published:2022-01-22
  • Contact: Fei Jun, MD, Associate chief physician, Department of Emergency, Army Specialty Medical Center, Army Medical University, Chongqing 400002, China
  • About author:Li Shidan, Master candidate, Attending physician, Department of Orthopedics, Army Specialty Medical Center, Army Medical University, Chongqing 400002, China
  • Supported by:
    the Special Fund for Improving Scientific and Technological Innovation Capacity of Army Medical University, No. 2019XLC2023 (to FJ); Chongqing Technology Innovation and Application Development Special General Project, No. cstc2019jscx-msxmX0210 (to LYB and WSC)

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摘要：

文题释义：
骨髓间充质干细胞：是一类起源于中胚层的成体干细胞，位于骨髓基质中，具有自我更新及多向分化潜能，同时还能分泌多种生长因子调节机体造血能力。1968年德国科学家FRIENDEN STEIN首次发现骨髓间充质干细胞，并进行体外培养，而后骨髓间充质干细胞逐渐成为研究热点。
国际细胞和基因治疗学会：1992年成立，由全球60多个国家和地区成员组成，是一个专注于细胞和基因疗法的临床前和转化方面研究的全球学会。2006年规范了间充质干细胞的标准：①贴壁生长；②表达阳性表面标志物CD105、CD73、CD90，不表达CD45、CD34、CD14或CD11b、CD79a或CD19、HLA-DR；③具有成骨、成脂肪、成软骨等多向分化潜能。

背景：提取人骨髓间充质干细胞主要有全骨髓贴壁培养法、密度梯度离心法、流式细胞仪分选及免疫磁珠分选等方法。流式分选及磁珠分选等方法对设备及试剂要求高，应用局限。全骨髓贴壁培养法及密度梯度离心法较为常用，但仍有全骨髓贴壁法细胞长出速度较慢、密度梯度离心法所需离心时间较长等不足。若发现一种更简便、且能够短时间提取骨髓间充质干细胞的方法则可进一步提高实验效率。
目的：介绍一种新的提取人骨髓间充质干细胞的方法，并与传统方法进行比较，证明新方法的可行性及实用性。 
方法：将磷酸缓冲盐溶液与人骨髓组织等比例混匀后离心使骨髓组织分层，取白膜层提取人骨髓间充质干细胞，与全骨髓贴壁法提取的人骨髓间充质干细胞进行对比。采用流式细胞分析及诱导多向分化实验进行鉴定。通过细胞计数、结晶紫染色、细胞骨架染色、划痕实验、Transwell迁移实验、RT-PCR等方法，分析两组细胞的原代细胞数目、传代后细胞形态、大小、细胞骨架、表面标志物表达、多向分化潜能、集落形成、增殖、迁移及成骨分化能力。 
结果与结论：①离心后磷酸缓冲盐溶液能够使人骨髓分层，并能够提取出人骨髓间充质干细胞；磷酸缓冲盐溶液法所提取的原代细胞数目较全骨髓贴壁法多，培养体系中漂浮脂肪组织少，原代细胞长出速度更快；②两种方法所提取细胞均呈梭形，表达标志物CD90、CD105、CD73，不表达CD45、CD34、CD11b、CD19及HLA-DR，具有多向分化潜能，符合间充质干细胞标准；③两种方法所提取细胞在形态、大小、细胞骨架方面没有显著差异，均能形成集落，且具有迁移能力，诱导成骨分化后两组细胞均表达成骨细胞标志物；④结果表明，在特定条件下利用磷酸缓冲盐溶液提取人骨髓间充质干细胞具有可行性及实用性。

https://orcid.org/0000-0002-7943-9299 (费军) 

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

关键词: 间充质干细胞, 骨髓间充质干细胞, 磷酸缓冲盐溶液, 全骨髓贴壁, 细胞提取, 细胞鉴定

Abstract: BACKGROUND: The methods to extract human bone marrow mesenchymal stem cells, including whole bone marrow adherent culture, density gradient centrifugation, flow cytometry separation and immunomagnetic bead separation. Flow cytometry separation and magnetic bead separation are restricted on equipment and reagents, so they are limited to applied. The whole bone marrow adherent culture method and density gradient centrifugation method are commonly used; however, there are still some shortcomings, such as slow cell growth rate and long centrifugation time. Thus, the efficiency of the experiment will be further improved if a more convenient and short time extraction method of bone marrow mesenchymal stem cells is found. 
OBJECTIVE: To introduce a new method of extracting human bone marrow mesenchymal stem cells and compare the extracted cells with those extracted by traditional methods to prove the feasibility and practicability of the new method.  
METHODS: Phosphate buffered saline was mixed with human bone marrow tissue at the same ratio, and the mixture was stratified by centrifugation. Human bone marrow mesenchymal stem cells were extracted from the white membrane layer followed by comparison with the cells extracted using whole bone marrow adherent method. The extracted cells were identified as bone marrow mesenchymal stem cells by flow cytometry and multidirectional differentiation potential. The number of primary cells, cell morphology, size, cytoskeleton, expression of surface markers, multidirectional differentiation potential, colony formation, proliferation, migration, and osteogenic differentiation were analyzed by cell counting, crystal violet staining, cytoskeleton staining, scratch test, Transwell migration test, and RT-PCR. 
RESULTS AND CONCLUSION: (1) After centrifugation, human bone marrow samples were stratified using phosphate buffered saline to extract human bone marrow mesenchymal stem cells. The number of primary cells extracted by phosphate buffered saline method was more than that of whole bone marrow adherent method. The floating adipose tissue was less in the culture system, and the primary cells grew faster. (2) The cells extracted by phosphate buffered saline and whole bone marrow adhesion were fusiform and expressed CD90, CD105, and CD73, but did not express CD45, CD34, CD11b, CD19, and HLA-DR. They had multidirectional differentiation potential and met the standard of mesenchymal stem cells. (3) There was no significant difference in morphology, size and cytoskeleton of the cells extracted using the two methods. Both groups of cells could form colonies and had migration ability. After osteogenic differentiation, both groups of cells expressed osteoblast markers. (4) The results show that it is feasible and practical to extract human bone marrow mesenchymal stem cells with phosphate buffered saline under certain conditions. 

Key words: mesenchymal stem cells, bone marrow mesenchymal stem cells, phosphate buffered saline, whole bone marrow adhesion, cell extraction, cell identification

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