中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (40): 7425-7429.doi: 10.3969/j.issn.1673-8225.2010.40.004

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

不同年龄儿童骨髓间充质干细胞自体血清体外培养及成骨分化

孙  客1，王国兵2，祖  莹2，唐盛平1，于  薇3，陈小文2   

  1. 深圳市儿童医院，1骨科，2儿研所，3超声科，广东省深圳市  518026
  • 出版日期:2010-10-01 发布日期:2010-10-01
  • 作者简介:孙客★，男，1971年生，四川省绵阳市人，汉族，2001年四川大学毕业，硕士，副主任医师，主要从事小儿骨科及矫形外科的研究。 soknow@126.com
  • 基金资助:

    深圳市科技项目(200803058)，课题名称：不同年龄儿童骨髓间充质干细胞培养及成骨分化的生物学特性研究。

Culture and osteogenic differentiation of different-aged children bone marrow mesenchymal stem cells in autologous serum in vitro

Sun Ke1, Wang Guo-bing2, Zu Ying2, Tang Sheng-ping1, Yu Wei3, Chen Xiao-wen2　   

  1. 1 Department of Orthopaedics, 2 Institute of Pediatric Surgery, 3 Department of Ultrasound, Shenzhen Children Hospital, Shenzhen  518026, Guangdong Province, China
  • Online:2010-10-01 Published:2010-10-01
  • About author:Sun Ke★, Master, Associate chief physician, Department of Orthopaedics, Shenzhen Children Hospital, Shenzhen 518026, Guangdong Province, China soknow@126.com
  • Supported by:

     the Science and Technology Program of Shenzhen City, No. 200803058*

PDF

354

被引次数

17

摘要：

背景：目前成人骨髓间充质干细胞体外培养成骨定向分化的研究较多，甚少见有不同年龄儿童骨髓间充质干细胞自体血清体外培养及成骨定向分化生物学特性差异的报道。
目的：研究不同年龄儿童骨髓间充质干细胞的自体血清体外培养条件与方法，并探讨其成骨诱导分化后的基本生物学特性。
方法：无菌条件下收集3个不同年龄组儿童骨髓悬液，分离人骨髓间充质干细胞，采用含自体血清的培养基培养；测定细胞生长曲线，采用流式细胞仪检测细胞表面抗原；取传代培养细胞，经β-甘油磷酸钠、抗坏血酸及地塞米松进行成骨诱导分化后，采用免疫组织化学法检测碱性磷酸酶、Ⅰ型胶原以及钙结节形成，以评价其分化效率；倒置相差显微镜逐日观察原代、传代及诱导分化细胞的生长情况及形态学改变。
结果与结论：3组儿童培养的人骨髓间充质干细胞，经刺激培养后细胞表面抗原CD29、CD44为阳性，CD34、CD45、CD105、CD106及HLA-DR均为阴性，各组表达强度差异无显著性意义。不同年龄组儿童人骨髓间充质干细胞生长速度以及分化效率随年龄增大而减小。结果提示通过对不同年龄组儿童人骨髓间充质干细胞生长特性、表面抗原表达以及分化潜能等方面的对比研究，自体血清可作为安全有效的培养方法，且年龄越小，分化效率相对越高。

关键词: 骨髓间充质干细胞, 儿童, 自体血清, 成骨分化, 细胞培养　

Abstract:

BACKGROUND: Most present studies emphasized on the induced osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) isolated from adults in vitro, but little was known about the osteogenic differentiation of hBMSCs isolated from children with different ages, or its biological characteristics after induced with autologous serum and osteogenic differentiation in vitro.
OBJECTIVE: To study culture conditions and methods of different-aged children BMSCs with autologous serum of in vitro culture, and explore its basic biological characteristics after osteogenic differentiation.
METHODS: Bone suspension of children, which were divided into three groups according to their age, was collected under aseptic conditions. hBMSCs were isolated and cultivated with media containing autologous serum. The cell growth curve was determined, and expression of cell surface antigens was analyzed by flow cytometry. After differentiation induced by β-sodium glycerophosphate, antiscorbic acid and dexamethasone, formation of alkaline phosphatase, type I collagen and calcium nodules of sub-cultivation cells were accessed by immunohistochemical method. The differentiation efficiency was evaluated. Growth conditions and morphological characteristics of primary cells, passage cells and differentiated cells were monitored using an inverted phase contrast microscope day by day.
RESULTS AND CONCLUSION: Among surface antigens cultivated hBMSCs from the three groups, CD29 and CD44 were detected to be positive, while CD34, CD45, CD105, CD106 and HLA-DR were found to be all negative. No significant difference of expression intensities were observed among three groups. The growth rate and differentiation efficiency of children BMSCs decreased with aging. Comparative studies on growth characteristics, surface antigen expression, differentiation potential of children hBMSCs suggested that autologous serum is a safe and effective culture method applicable to clinic with a high differentiation efficiency at the low child ages.

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