中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (6): 951-956.doi: 10.3969/j.issn.1673-8225.2010.06.001

• 干细胞培养与分化 •    下一篇

犬骨髓间充质干细胞分离纯化和成骨诱导分化：Ficoll液密度梯度离心法
体外分离的可行性

解  芳，滕  利，蔡  磊，徐家杰，靳小雷，肖  苒，曹谊林   

  1. 中国医学科学院/北京协和医学院，整形外科医院，北京市  100144
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 通讯作者: 通讯作者：滕 利，博士，教授，主任医师，博士生导师，中国医学科学院/北京协和医学院，整形外科医院，北京市 100144 tenglidr@sina.com 曹谊林，博士，教授，中国医学科学院/北京协和医学院，整形外科医院，北京市 100144 yilincao@yahoo.com
  • 作者简介:解 芳，女，1984年生，山西省太原市人，汉族，北京协和医学院在读硕士，主要从事骨组织工程方面的研究。 cristina_jackson 2000@yahoo.com.cn
  • 基金资助:

    卫生部临床学科重点项目(3030426-04)

Isolation, purification and osteoinduction differentiation of canine bone marrow mesenchymal stem cells: Feasibility of in vitro isolation using Ficoll density gradient centrifugation

Xie Fang, Teng Li, Cai Lei, Xu Jia-jie, Jin Xiao-lei, Xiao Ran, Cao Yi-lin   

  1. Plastic Surgery Hospital, Peking Union Medical College & Chinese Academy of Medical Science, Beijing   100144, China
  • Online:2010-02-05 Published:2010-02-05
  • Contact: Teng Li, Doctor, Professor, Chief physician, Doctoral supervisor, Plastic Surgery Hospital, Peking Union Medical College & Chinese Academy of Medical Science, Beijing 100144, China tenglidr@sina.com Cao Yi-lin, Doctor, Professor, Plastic Surgery Hospital, Peking Union Medical College & Chinese Academy of Medical Science, Beijing 100144, China yilincao@yahoo.com
  • About author:Xie Fang, Studying for master’s degree, Plastic Surgery Hospital, Peking Union Medical College & Chinese Academy of Medical Science, Beijing 100144, China
  • Supported by:

    the Clinical Speciality Key Program of Public Health Ministry of China, No. 3030426-04*

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摘要：

背景：目前骨髓间充质干细胞较经典的分离方法是Percoll密度梯度离心法，以去除血细胞成分，但该法操作较为复杂，分离犬骨髓时需要配制密度，且离心次数较多，对细胞损伤大。

目的：拟建立可靠、高纯度的犬骨髓间充质干细胞体外分离纯化方法，并观察其在体外扩增、成骨诱导分化的能力。

方法：于犬髂后上棘抽取骨髓液10 mL，肝素抗凝，Hanks液稀释后，加入1.077 g/mL Ficoll液3 mL，2 000 r/min离心20 min，吸取有核细胞形成白色云雾状的分层界面，用含胎牛血清的DMEM离心2遍，按12×104/cm2密度接种，于37 ℃、体积分数为5%的CO2培养箱内培养。细胞传代后，加入含地塞米松、β-磷酸甘油钠、L-2-磷酸抗坏血酸的DMEM成骨条件培养液进行诱导。免疫细胞化学染色和免疫荧光染色检测成骨细胞特征性分泌蛋白骨钙素、骨桥蛋白的表达，以及Ⅰ型胶原的表达，并进行碱性磷酸酶染色与茜素红染色。

结果与结论：1.077 g/mL Ficoll液密度梯度离心法分离所得的有核细胞层相对于Percoll液分层明显，可获得纯度较高的骨髓间充质干细胞，接种细胞生长良好，平均倍增时间为24 h。犬骨髓间充质干细胞体外成骨诱导培养后，骨钙素、骨桥蛋白、Ⅰ型胶原均呈阳性表达，碱性磷酸酶染色后细胞胞浆呈蓝绿色，茜素红染色后细胞外基质中出现散在的红色结节，可在体外定向分化为成骨细胞。

关键词: 成骨诱导, Ficoll, 密度梯度离心, 犬, 骨髓间充质干细胞, 干细胞

Abstract:

BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells.

OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation.

METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×104/cm2 at 37 ℃ in a 5% CO2 incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed.

RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.

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