中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (2): 191-195.doi: 10.3969/j.issn.1673-8225.2010.02.001

• 骨组织构建 bone tissue construction •    下一篇

巨噬细胞集落刺激因子/核因子κB受体激活物配体体外诱导培养高纯度破骨细胞：最佳剂量探讨

包洪卫1，孙继芾2，王  青2   

  1. 1靖江市人民医院骨科，江苏省靖江市  214500；2南京医科大学第一附属医院骨科，江苏省南京市 210029
  • 出版日期:2010-01-08 发布日期:2010-01-08
  • 通讯作者: 王 青，副教授，南京医科大学第一附属医院骨科，江苏省南京市 210029 dr.wangqing@163.com
  • 作者简介:包洪卫，男，1968年生，江苏省靖江市人，汉族，1991年南京医科大学毕业，副主任医师，主要从事关节外科研究。 jjbhw@126.com
  • 基金资助:

    江苏省卫生厅课题资助项目(H200601)。

In vitro culture of high-purity osteoclasts induced by macrophage colony stimulating factor/receptor activator of nuclear factor kappa B ligand: Optimal dosage investigation

Bao Hong-wei1, Sun Ji-fu2, Wang Qing2   

  1. 1 Department of Orthopaedics, Jingjiang People’s Hospital, Jingjiang   214500, Jiangsu Province, China; 2 Department of Orthopaedics, First Affiliated Hospital of Nanjing Medical University, Nanjing   210029, Jiangsu Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Wang Qing, Associate professor, Department of Orthopaedics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China dr.wangqing@163.com
  • About author:Bao Hong-wei, Associate chief physician, Department of Orthopaedics, Jingjiang People’s Hospital, Jingjiang 214500, Jiangsu Province, China jjbhw@126.com
  • Supported by:

    a grant by Jiangsu Provincial Health Bureau, No. H200601* 

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摘要：

背景：巨噬细胞集落刺激因子(macrophage colony-stlimulating factor，M-CSF)/核因子κB受体激活物配体(receptor activator of nuclear kappa B ligand，RANKL)两种细胞因子协同诱导骨髓干细胞形成破骨细胞是一种较新的，可以获取较高纯度和数量破骨细胞的诱导培养法，但尚缺乏统一的培养标准。
目的：建立有效的M-CSF/RANKL诱导小鼠骨髓干细胞诱导分化破骨细胞的培养体系。
方法:分离小鼠四肢骨获取骨髓干细胞。将小鼠骨髓干细胞在含有M-CSF的α-MEM培养基中培养24 h，调整细胞浓度为107，108，109 L-1。然后在培养基中同时加入10 µg/LM-CSF和不同质量浓度(20，50，100 µg/L)RANKL。行抗酒石酸酸性磷酸酶染色观察干细胞向破骨细胞的转变过程及细胞形态和染色情况，并对各组染色阳性的破骨细胞进行计数，比较不同诱导条件对破骨样细胞数量的影响。
结果与结论：培养3 d后可见少量破骨样细胞，胞质内含许多红色抗酒石酸酸性磷酸酶染色阳性颗粒，细胞内可见淡染的双核；培养6 d后可见大量染色阳性破骨样细胞；培养9 d后出现多核巨型染色阳性破骨样细胞，细胞明显增大，细胞核可达到3个以上。在固定细胞接种浓度条件下，RANKL质量浓度为100 µg/L时诱导分化形成的破骨样细胞数量较另两质量浓度增多(P < 0.05)；在固定RANKL质量浓度条件下，细胞浓度为108 L-1时诱导分化形成的破骨样细胞数量较另两细胞接种浓度增多(P < 0.05)；细胞接种浓度为108 L-1, RANKL质量浓度为100 µg/L时诱导分化形成的破骨样细胞数量高于其他条件组合(P < 0.05)。说明在M-CSF/RANKL诱导小鼠骨髓干细胞分化培养破骨细胞的体系中，RANKL最佳质量浓度为 100 µg/L，最佳细胞接种浓度为108 L-1。

关键词: M-CSF, RANKL, 破骨细胞, 骨髓干细胞, 骨组织工程

Abstract:

BACKGROUND: Macrophage colony-stimulating factor (M-CSF)/receptor activator of nuclear kappa B ligand (RANKL), two types of cytokines co-induce myeloid stem cells to form osteoclasts, is a kind of new method to harvest osteoclasts with high purity and quantity, but there is lack of uniform cultivation standard.
OBJECTIVE: To construct an effective M-CSF/RANKL induced mice myeloid stem cells inducing osteoclast differentiation cultivation system.
METHODS: Myeloid stem cells were obtained from ICR mice and then cultured for 24 hours in a-minimum essential medium containing M-CSF, at cell density of 107/L, 108/L, 109/L. Then 10 µg/L M-CSF and 20, 50, 100 µg/L RANKL were added into culture medium. Tartaric-resistant acid phosphatase stained was performed to observe the transition process from stem cell to osteoclast, as well as cell morphology and stain situation after culture, and positive stained osteoclasts were counted. We compared the influence of different induction conditions to the quantity of osteoclast.
RESULTS AND CONCLUSION: A small quantity of osteoclasts contained many red positive beads in the intracytoplasm were observed at 3 days. There were positive beads with hypochromatic dikaryon in cells. A large amount of positively stained osteoclasts were seen after 6-day culturing, which maintained dikaryon. After 9-day culturing, positively stained colossal multinuclear cells occurred, became larger and maintained three nuclei. At certain cell density, 100 µg/L RANKL could induce to form more osteoclasts compared with other 2 concentrations (P < 0.05); at certain RANKL concentration, the osteoclasts formation at cells density of 108/L was dramatically greater than other 2 cell densities (P < 0.05); the number of osteoclasts was the most when the concentration of RANKL was 100 µg/L and cell density of 108/L (P < 0.05). When osteoclasts are induced by M-CSF/RANKL from murine myeloid stem cells, the best concentration of RANKL is 100 µg/L and cells density is 108/L.

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