Characterization of circulating human osteoclast progenitors:: Development of in vitro resorption assay
Characterization of circulating human osteoclast progenitors:: Development of in vitro resorption assay
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DOI：
10.1007/s00223-004-0123-z
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发表时间：
2005-03-01
影响因子：
4.2
通讯作者：
Hentunen, TA
中科院分区：
文献类型：
--
作者：
Husheem, M;Nyman, JKE;Hentunen, TA
采用免疫磁性细胞分离系统，用多种细胞表面标志分离单核细胞作为破骨祖细胞。将该系统与特异性单核细胞抗体结合使用，可获得99%的纯单核细胞。纯化的单核细胞在含有核因子受体激活剂-kappa B(RANKL)、巨噬细胞集落刺激因子(M-CSF)、肿瘤坏死因子-α(TNF-α)和地塞米松的条件下培养14天后，CD14(+)、CD11b(+)和CD61(+)单核细胞的破骨细胞形成能力分别是对照组的90、30和20倍。CD15(+)单核细胞很少产生抗酒石酸酸性磷酸酶阳性的多核细胞(TRACP+MNC)，CD169(+)单核细胞不产生TRACP+MNC。这表明，血液循环中存在不同的单核细胞亚群，它们在破骨细胞形成方面具有不同的能力。这些结果表明，使用抗CD14、-CD11b和-CD61抗体的免疫磁性细胞分离系统可以有效地纯化循环中的人破骨细胞前体细胞。这些纯化的单核细胞组分具有不同的诱导破骨细胞的能力。CD169不适用于破骨细胞前体细胞的分离。破骨细胞形成和骨吸收的最佳地塞米松浓度为10 nM。为建立人骨吸收实验方法，首先诱导破骨细胞7d，更换整个培养液，继续培养3d，从培养液中测定I型胶原的C末端端肽。由于两种广为人知的吸收抑制剂，巴菲尔霉素A(1)和降钙素，对破骨细胞的吸收活性有剂量依赖性的抑制作用，这种方法被证明是有效的。
Several cell surface markers were used to isolate monocytes as osteoclast progenitors with an immunomagnetic cell separation system. Use of this system with specific monocyte antibodies produced 99% pure monocytes. When purified monocytes were cultured on bovine bone slices in the presence of receptor activator of nuclear factor-kappa B (RANKL), macrophagecolony stimulating factor (M-CSF), tumor necrocis factor alpha (TNF-alpha), and dexamethasone for 14 days, CD14(+) CD11b(+), and CD61(+) monocytes had approximately 90-, 30- and 20-fold higher osteoclast formation capacities/plated cells compared to the control culture. CD15(+) monocytes generated few tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNC), and CD169(+) monocytes generated no TRACP+ MNC. This suggests, that there are various subsets of monocytes in the blood circulation and that they have different capacities in osteoclast formation. These results show that circulating human osteoclast progenitors can be efficiently purified by immunomagnetic cell separation system using anti-CD14, -CD11b, and -CD61 antibodies. These purified monocyte fractions had different ability to give rise to osteoclasts. CD169 was not found to be Suitable for osteoclast progenitor isolation. Optimal concentration of dexamethasone for osteoclast formation and bone resorption was 10 nM. To develop a human resorption assay, osteoclasts were first induced for 7 days, whole media were replaced, cultures were continued for additional 3 days and C-terminal telopeptide of type I collagen was determined from culture media. This assay was shown to be functional, since two well-known resorption inhibitors, bafilomycin A(1) and calcitonin, dose-dependently inhibited the resorption activity of osteoclasts.