中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (27): 4984-4987.doi: 10.3969/j.issn.1673-8225.2010.27.010

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

脐血间充质干细胞生物学特性及其成骨成脂分化潜能

张积华1，孙  康2，王  妍3，田少奇2，夏长所2，张才龙2，于腾波2   

  1. 青岛大学医学院附属医院，1查体中心，2关节外科，山东省青岛市  266003；3青岛市妇女儿童医院，山东省青岛市  266000
  • 出版日期:2010-07-02 发布日期:2010-07-02
  • 通讯作者: 孙 康，教授，青岛大学医学院附属医院关节外科，山东省青岛市 266003 Sunkang_qy@ yahoo.com.cn
  • 作者简介:张积华，女，1959年生，山东省蓬莱市人，汉族，2006年滨州医学院毕业，主要从事干细胞基础与超声诊断学方面的研究。 qingyiren66@ 163.com
  • 基金资助:

    山东省科技攻关计划(2008GG30002037)。

Biological characteristics, osteogenic and adipogenic differentiation potential of mesenchymal stem cells from human umbilical cord blood

Zhang Ji-hua1, Sun Kang2, Wang Yan3, Tian Shao-qi2, Xia Chang-suo2, Zhang Cai-long2, Yu Teng-bo2   

  1. 1 Physical Examination Center, 2 Department of Orthopaedics, Affiliated Hospital of Medical College, Qingdao University, Qingdao  266003, Shandong Province, China; 3 Qingdao Woman and Children’s Hospital, Qingdao  266000, Shandong Province, China
  • Online:2010-07-02 Published:2010-07-02
  • Contact: Sun Kang, Professor, Department of Orthopaedics, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China Sunkang_qy@yahoo.com.cn
  • About author:Zhang Ji-hua, Physical Examination Center, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, Shandong Province, China qingyiren66@163. com
  • Supported by:

    the Key Project in Science and Technology of Shandong Province, No. 2008GG30002037*

PDF

399

被引次数

17

摘要：

背景：对于脐血来源间充质干细胞的研究是干细胞研究领域的重点和热点，但目前国内外研究对其报道尚较少，许多方面存在争议。
目的：观察人脐血间充质干细胞的生物学特性，并探讨其向成骨、成脂肪细胞诱导分化的能力。
方法：从不同胎龄脐血中分离培养间充质干细胞；通过倒置相差显微镜及透射电镜观察细胞的形态、生长增殖情况及其超微结构；并描绘细胞生长曲线；用流式细胞仪对细胞表面标志物进行检测。分别用成骨及成脂诱导液对脐血间充质干细胞进行诱导，通过茜素红染色检测脐血间充质干细胞向成骨细胞诱导分化的能力；通过油红O染色检测其向脂肪细胞诱导分化的能力。
结果与结论：倒置相差显微镜下观察脐血间充质干细胞贴壁生长，呈成纤维细胞样外观，细胞呈螺旋状排列；透射电镜观察脐血间充质干细胞细胞核大，胞核比例大，细胞器少，为低分化细胞；原代及传代培养的脐血间充质干细胞生长曲线均呈S型，第3，5代细胞增殖能力最强。流式细胞仪检测结果显示，脐血间充质干细胞均稳定表达与间充质干细胞相关的表面抗原标志物CD29，CD44和CD90等，不表达造血标志CD34和CD45。脐血间充质干细胞成骨诱导后3周时茜素红染色可检测到大量钙化基质的形成；成脂诱导3周时油红O染色可检测到胞质中脂滴的形成。提示脐血间充质干细胞的形态特征、生长增殖特点及细胞表面标志物等生物学特性与骨髓间充质干细胞相似，具有强大的生长增殖与自我更新能力。脐血间充质干细胞在体外诱导条件下可以向成骨细胞及脂肪细胞等间质组织细胞定向分化。

关键词: 脐血, 间充质干细胞, 生物学特性, 成骨细胞, 成脂肪细胞

Abstract:

BACKGROUND: Studies addressing umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are key point and hot focus in the research of stem cells. However, few reports have addressed UCB-MSCs, and there are controversial in many aspects.
OBJECTIVE: To observe the biological characteristics, osteogenic and adipogenic differentiation potential of UCB-MSCs.
METHODS: MSCs were harvested and cultured from UCB at various gestational ages. The morphology and ultramicrostructure of MSCs and their growth characteristics in vitro were observed under an inverted phase contrast microscope and transmission electron microscope. The surface antigen phenotype was analyzed by flow cytometry. The osteogenic and adipogenic media were used to induce differentiation of UCB-MSCs into osteoblasts and adipocytes. The osteogenic potential was determined by alizarin red staining. Adipogenic potential was detected by oil-red O staining.
RESULTS AND CONCLUSION: Under the inverted phase contrast microscope, UCB-MSCs were adherent with a fibroblast like morphology and whirlpool like growth alinement. The ultramicrostructure in transmission electron microscope showed that UCB-MSCs had a big cell nucleus, less cellular organelles and big karyoplasmic ratio. All of the growth curves of primary and subculturing UCB-MSCs presented “S” type. The third and fifth generation of MSCs showed the greatest reproductive activity. And the count of CFU-F was higher in smaller gestational age than the older. Flow cytometry showed that these cells were positive for CD29, CD44 and CD90 expression, but they failed to express hematopoictic cell surface markers, such as CD34 and CD45. When the MSCs were induced to osteogenic and adipogenic differentiation for 3 weeks, the formation of a mineral extracellular matrix and neutral lipid vacuoles detected by alkaline phosphatase staining, alizarin red staining and oil-red O staining respectively. Results have indicated that UCB-MSCs have similar morphological character, biological characteristics and cell surface markers with MSCs derived from bone marrow, both of which have great capability of proliferation and regeneration. UCB-MSCs can be induced to differentiation to osteoblasts and lipoblasts in a suitable condition in vitro.

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