人外周血CD68+单核细胞体外的分选及诱导分化为破骨细胞

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (05): 585-588.

人外周血CD68⁺单核细胞体外的分选及诱导分化为破骨细胞

曾润铭, 金大地, 张忠民

第一军医大学南方医院脊柱骨科, 广东, 广州, 510515

出版日期:2004-05-20 发布日期:2004-05-20
基金资助:
收稿日期:2004-1-11。
作者简介:曾润铭(1967- ),男,1989年毕业于汕头大学医学院,主冶医生,现为第一军医大学在读硕士研究生,电话:020-61641725,E-mail:hosward@21cn.com

Induced differentiation of human peripheral blood CD68⁺ mononuclear cells into osteoclasts in vitro

ZENG Run-ming, JIN Da-di, ZHANG Zhong-min

第一军医大学南方医院脊柱骨科, 广东, 广州, 510515

Online:2004-05-20 Published:2004-05-20

摘要/Abstract

摘要： 目的建立高纯度人活性破骨细胞方法,用于对破骨细胞生物化学和分子生物学的研究。方法用免疫磁珠法在人类外周血单核细胞中分选出CD68⁺细胞,用流式细胞仪分析分选效能,体外在10^-8 mol/L地塞米松及25 μg/L巨噬细胞集落刺激因子的培养条件下用16 μg/L可溶性核因子κB受体活化子配体诱导(s-RANKL)分化,用降钙素受体抗体免疫组化染色及抗酒石酸磷酸酶染色鉴定,扫描电镜观察骨片贴壁细胞及骨吸收陷窝。结果分选获得CD68⁺单核细胞纯度为(93.06±0.61)%(n=4),经核因子κB受体活化子配体诱导后降钙素受体抗体免疫组化染色阳性,抗酒石酸磷酸酶染色,扫描电镜观察有骨吸收陷窝形成。结论人外周血单核细胞中CD68⁺细胞是破骨前体细胞,在RANKL在诱导下分化为成熟破骨细胞。

Abstract: Objective To establish a method for obtaining highly purified primary human osteoclast precursors for the biochemical and molecular biological research. Methods CD68⁺ mono/macrophages were separated from peripheral blood mononuclear cells (PBMCs) of healthy donors by means of immunomagnetic cell sorting for subsequent analysis with flow cytometry. The isolated cells were incubated on coverslips or bone slices in the presence of dexamethasone(10^-8 mol/L), macrophage colony-stimulating factor (25 μg/L ) and soluble receptor activator of nuclear factor (NF)-κB ligand (s-RANKL, 16 μg/L). Calcitonin receptor (CR) immunocytochemistry and tartrate-resistant acid phosphatase (TRAP) histochemistry were employed. The bone slices were also studied by scanning electron microscopy (SEM). Results Fluorescence-activated cytometric analysis showed that 93.06%±0.61%(n=4)of the selected cells were CD68⁺ cells. After 7 days of culture of the CD68⁺ cells, VR+, TRAP+ multinucleated giant cells appeared, and resorption lacunae could be observed by SEM. Conclusion Highly purified CD68⁺ cells can be obtained from human PBMCs as the osteoclast precursors, and mature osteoclasts can be induced from CD68⁺ mono/macrophages by RANKL.

中图分类号:

R392.11

曾润铭, 金大地, 张忠民. 人外周血CD68⁺单核细胞体外的分选及诱导分化为破骨细胞[J]. 南方医科大学学报, 2004, 24(05): 585-588.

ZENG Run-ming, JIN Da-di, ZHANG Zhong-min. Induced differentiation of human peripheral blood CD68⁺ mononuclear cells into osteoclasts in vitro[J]. Journal of Southern Medical University, 2004, 24(05): 585-588.

参考文献

[1] Shinoda K, Sugiyama E, Taki H, et al. Resting T cells negatively regulate osteoclast generation from peripheral blood monocytes [J].Bone, 2003, 33(4): 711-20.
[2] Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis[J]. Nature, 1999, 397: 315-23.
[3] Choi Y, Woo K M, Ko S H, et al. Osteoclastogenesis enhanced by activated B cells but suppressed by CD8+ T cells [J]. Eur J Immunol, 2001, 31: 2179-88.
[4] Weitzmann MN, Cenci S, Haug J, et al. B lymphocytes inhibit human osteoclastogenesis by secretion of TGF-beta [J]. J Cell Biochem, 2000, 78(2): 318-24.
[5] Julian M W, Quinn JM, Elliott J, et al. A combination ofosteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro [J]. Endocrinology, 1998, 139(10): 4424-7.
[6] Nicholson GC, Malakellis M, Collier FM, et al. Induction ofosteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL)[J]. Clin Sci (Lond), 2000 Aug, 99(2): 133-40.
[7] Shalhoub V, Elliott G, Chiu L, et al. Characterization of osteoclast precursors in human blood [J]. Br J Haematol, 2000, 111 (2):501-12.
[8] Athanasou NA, Quinn J. Immunophenotypic differences between osteoclasts and macrophage polykaryons: immunohistological distinction and implications for osteoclast ontogeny and function[J]. J Clin Pathol, 1990, 43: 997-1003.
[9] Fiorentini S, Licenziati S, Alessandri G, et al. CD1 lb expression identifies CD8+CD28+ T lymphocytes with phenotype and function of both naive/memory and effeetor cells[J]. J Immunol, 2001, 166(2):900-7.
[10] Christensen JE, Andreasen SO, Christensen JP, et al. CD1 lb expression as a marker to distinguish between recently activated effector CD8+ T cells and memory cells [J]. Int Immunol, 2001, 13 (4):593-600.
[11] Kong Y Y, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand[J]. Nature, 1999, 402(18): 304-9.
[12] 蒋建平,杨铁城,侯凡凡,等.人类关节滑膜A型和B型细胞的分离和体外培养[J].第一军医大学学报,2001,21(1):52-5.Jiang JP, Yang TC, Hou FF, et al. Isolation and culture of human joint type-A and type-B synovial cells. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2001, 21(1):52-5.