中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (10): 1742-1747.doi: 10.3969/j.issn.1673-8225.2012.10.008

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

人骨髓基质干细胞的单细胞克隆培养与鉴定***☆

滕  勇1，徐建丽2，李旭升1，胡蕴玉3，王  臻3   

  1. 1解放军兰州军区乌鲁木齐总医院骨一科，新疆维吾尔自治区乌鲁木齐市  830000；  2新疆医科大学第一附属医院血液科，新疆维吾尔自治区乌鲁木齐市  830000；3解放军第四军医大学西京医院骨科研究所，陕西省西安市  710032
  • 收稿日期:2011-09-24 修回日期:2012-02-01 出版日期:2012-03-04 发布日期:2012-03-04
  • 作者简介:滕勇☆，男，1972年出生，重庆市人，汉族，2005年解放军第四军医大学毕业，博士，副主任医师，硕士生导师，主要从事骨组织工程、骨缺损修复方面的研究。orthtengyong@163.com
  • 基金资助:

    解放军兰州军区医药卫生科研计划项目(LXH2007014)；国家自然科学基金资助项目(51165044)；全军十二五课题基金资助项目(CWS11J011)

Single-cell clonal culture and identification of human bone marrow stromal stem cells  

Teng Yong1, Xu Jian-li2, Li Xu-sheng1, Hu Yun-yu3, Wang Zhen3   

  1. 1First Department of Orthopedics, Urumqi General Hospital of Lanzhou Command, PLA, Urumqi 830000, Xinjiang Uygur Autonomous Region, China; 2Department of Hematology, First Affiliated Hospital of Xinjiang Medical University, Urumqi  830000, Xinjiang Uygur Autonomous Region, China;      3Institute of Orthopedics, Xijing Hospital, Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China
  • Received:2011-09-24 Revised:2012-02-01 Online:2012-03-04 Published:2012-03-04
  • About author:Teng Yong☆, M.D., Associate chief physician, Master’s supervisor, First Department of Orthopedics, Urumqi General Hospital of Lanzhou Command, PLA, Urumqi 830000, Xinjiang Uygur Autonomous Region, China orthtengyong@163.com
  • Supported by:

    Medical Science and Research Development of Lanzhou Command, No.LXH2007014*; the National Natural Science Foundation of China, No.51165044*; a grant from the Twelfth “Five-Year” Development Program Foundation of Chinese PLA, No.CWS11J01*

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摘要：

背景：骨髓基质干细胞缺乏特异的表面识别分子，其鉴定一直是研究中的难题。
目的：探索人骨髓基质干细胞培养条件，获得体外克隆化培养的成人骨髓基质干细胞，并对其进行表型分析及分化潜能鉴定。
方法：外科手术取髂骨术中抽取骨髓，采用密度梯度离心法初步分离骨髓基质干细胞，用极限稀释法进行克隆化培养；流式细胞仪对克隆化培养的骨髓基质干细胞进行细胞表面标志检测，并进行体外成软骨、成骨及心肌细胞诱导，免疫组织化学及RT-PCR检测成软骨、成骨及心肌细胞表达，确定其表型及分化潜能。
结果与结论：单个细胞来源骨髓基质干细胞在体外1∶3传代一般可以传28代左右，24代以前生长状态良好；经流式细胞仪检测，骨髓基质干细胞表达CD29，CD44，CD106，不表达CD14，CD34，CD45，HLA-DR；骨髓基质干细胞体外可向成骨、成软骨及心肌细胞分化，提示体外克隆化培养的骨髓基质干细胞能维持良好的成体干细胞生物学特性。
关键词：单克隆培养；骨髓基质干细胞；多能成体干细胞；鉴定；分化
doi:10.3969/j.issn.1673-8225.2012.10.008

关键词: 单克隆培养, 骨髓基质干细胞, 多能成体干细胞, 鉴定, 分化

Abstract:

BACKGROUND: Bone marrow stromal stem cells lack of specific surface marker and their identification challenges scholars all the time.
OBJECTIVE: To explore the culture condition for clonal isolation of human bone marrow stromal stem cells (BMSCs) and identify their surface markers and differentiation potentials.
METHODS: Human bone marrow was taken and BMSCs were isolated using density gradient centrifugation. Clone-like cells were selected by single cell limiting dilution. The surface antigens of the cloned BMSCs were analyzed by flow cytometry and immunocytochemistry. Multi-differentiation capacities were evaluated by inducing BMSCs into osteoblasts, chondrocytes and cadiocytes. RT-PCR and immunocytochemistry were applied to detect the three mutli-differentiations.
RESULTS AND CONCLUSION: Single cell-derived BMSCs could be expanded to passage 28 in vitro which still maintaued active proliferation ability.The expanded BMSCs expressed CD29, CD44, CD106, but not CD14, CD34, CD45, HLA-DR. They could be induced to differentiate into osteoblasts, chondrocytes and cadiocytes. These findings suggest that cloned BMSCs can maintain the phenotypes of stem cells during in vitro culture.

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