中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (6): 1002-1005.doi: 10.3969/j.issn.1673-8225.2010.06.010

• 干细胞培养与分化 • 上一篇    下一篇

兔骨髓间充质干细胞体外分离培养及多向诱导分化

徐成峰，胡大海，赵周婷，张万福，白晓智，蔡维霞   

  1. 解放军第四军医大学西京医院烧伤与皮肤外科，全军烧伤中心，陕西省西安市  710032
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 通讯作者: 胡大海，教授，主任医师，博士生导师，解放军第四军医大学西京医院烧伤与皮肤外科，全军烧伤中心，陕西省西安市 710032
  • 作者简介:徐成峰，男，1983年生，湖北省枣阳市人，汉族，解放军第四军医大学在读硕士，医师，主要从事创面及组织修复方向的研究。 xcf1983@yahoo.com.cn
  • 基金资助:

    国家自然科学基金(30772249)

Isolation, culture and multiple differentiations of rabbit bone marrow-derived mesenchymal stem cells in vitro

Xu Cheng-feng, Hu Da-hai, Zhao Zhou-ting, Zhang Wan-fu, Bai Xiao-zhi, Cai Wei-xia   

  1. Department of Burns and Cutaneous Surgery, Burns Center of PLA, Xijing Hospital, the Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China
  • Online:2010-02-05 Published:2010-02-05
  • Contact: Hu Da-hai, Professor, Chief physician, Doctoral supervisor, Department of Burns and Cutaneous Surgery, Burns Center of PLA, Xijing Hospital, the Fourth Military Medical Universityof Chinese PLA, Xi’an 710032, Shaanxi Province, China
  • About author:Xu Cheng-feng, Studying for master’s degree, Physician, Department of Burns and Cutaneous Surgery, Burns Center of PLA, Xijing Hospital, the Fourth Military Medical University of Chinese PLA, Xi’an 710032, Shaanxi Province, China xcf1983@yahoo.com.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30772249*

PDF

509

被引次数

33

摘要：

背景：间充质干细胞不仅自身免疫原性弱，还可以调节细胞免疫功能，减轻移植物排斥反应，在组织工程中具有良好的应用前景。但骨髓中间充质干细胞含量稀少，约占单个核细胞的十万分之一到百万分之一。

目的：建立一种分离、培养扩增兔骨髓间充质干细胞的方法，观察体外培养骨髓间充质干细胞的生长特性，及其潜在的诱导分化能力。

方法：采用灌流法获取兔胫骨骨髓，密度梯度离心法联合贴壁培养法体外纯化扩增，相差显微镜观察其形态学特点，MTT法测定绘制传代骨髓间充质干细胞生长曲线，经成骨诱导液(L-DMEN/F12，体积分数为10%胎牛血清，0.1 μmol/L地塞米松，200 μmol/L抗坏血酸，10 mmol/L β-甘油磷酸钠)、成脂诱导液(L-DMEN/F12，体积分数为10%胎牛血清，1 μmol/L地塞米松，200 μmol/L吲哚美辛，0.5 mmol/L IBMX，10 mg/L胰岛素)、成软骨诱导液(L-DMEN/F12，体积分数为10%胎牛血清，10 μg/L转化生长因子β1，0.1 μmol/L地塞米松，50 μmol/L抗坏血酸，6.25 mg/L胰岛素)体外诱导骨髓间充质干细胞向成骨细胞、脂肪细胞及软骨细胞分化，并分别经碱性磷酸酶染色、油红O染色和甲苯胺蓝染色法鉴定。

结果与结论：通过密度梯度离心法联合贴壁培养法可在体外大量扩增、纯化骨髓间充质干细胞，所获细胞具有高度自我更新能力和多向分化潜能，原代及传代骨髓间充质干细胞为梭形。经成骨细胞诱导，细胞碱性磷酸酶染色阳性；经成脂肪细胞诱导，细胞内出现红色脂滴，经成软骨细胞诱导，甲苯胺蓝染色阳性。

关键词: 间充质干细胞, 细胞培养, 分化, 兔, 骨髓间充质干细胞

Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs), with low immunogenicity, can regulate cellular immunity and mitigate graft rejection, which has a good prospect in tissue engineering. However, it is rarely present in bone marrow. 

OBJECTIVE: To explore an isolation and culture method of the rabbit bone marrow-derived MSCs, to observe the biological characteristics and differentiation potential of bone marrow-derived MSCs.

METHODS: MSCs were isolated from rabbit tibia bone marrow by combination of gradient centrifugation and different adherent method, then proliferation in vitro. Morphology was examined by phase contrast microscopy, and the growth curve of cultured MSCs was drawn via MTT results. MSCs were treated with osteogenetic inductor (L-DMEM/F12, 10% fetal bovine serum, 0.1 μmol/L dexamethasone, 200 μmol/L vitamin C, 10 mmol/L β-phosphoglycerol), adipose inductor (L-DMEM/F12, 10% FBS, 1 μmol/L dexamethasone, 200 μmol/L antifani, 0.5 mmol/L IBMX, 10 μg/mL insulin), and chondrocytes inductor (L-DMEM/F12, 10% FBS, 10 μg/L TGF-β1, 0.1 μmol/L dexamethasone, 50 μmol/L vitamin C, 6.25 mg/L insulin) to differentiated into osteoblast, dipocytes and chondrocytes. And the differentiated cells were identified by alkaline phosphatase staining, oil red O staining, and toluidine blue staining, respectively.

RESULTS AND CONCLUSION: Bone marrow-derived MSCs can be isolated and cultured by the combination of gradient centrifugation and different adherent method in vitro, which have the better potentiality of proliferation and multi-directional differentiation. Mostly of the primary and passaged cells were spindle-shaped. After osteogenetic induction, cells were positive to alkaline phosphatase staining. Oil red O staining showed that red lipid droplet existed in adipose cells, and toluidine blue staining showed that toluidine blue was positive after chondrocytes induction.

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