Zhu Mei, Zhang Peng, Zhang Xin, Lu Biao, Dai Chen-lin, Li Yu-min, Qiu Ming-cai
Department of Endocrinology, General Hospital of Tianjin Medical University, Tianjin 300052, China.
Zhonghua Yi Xue Za Zhi. 2005 Mar 23;85(11):738-42.
To investigate the effects of osteoclast-like cells (OLC) and its sub-cellular structures on the osteoblast (OB) differentiation and function.
Spleen cells from C57 mice administrated with 5-fluorouracil were induced by IL-3, 6 and granulocyte-macrophage colony stimulating factor (GM-CSF), and 1alpha, 25-(OH)(2)D(3) to obtain massive OLCs. These OLC cells were cultured in culture fluid and on bone wafers (called bolcs). Osteoblasts were cultured and added with NaF, OLCs of two kinds, culture fluid free of OLC, and sub-unit structures such as nucleus, mitochondria, and cytoplasma from OLCs for 5 days. The proliferation rate of OBs was measured by MTT method and the alkaline phophatase (ALP) activity was measured by PNPP method. Immunochemistry was used to detect the core-binding factor alpha1 (Cbfalpha1) in the OBs, Enzyme linked immunosorbent assay was used to measure the osteocalcin.
The OB number was lower in the OLC (1.288 +/- 0.039), OLC cytoplasm (1.138 +/- 0.024), 50% OLC culture fluid (1.203 +/- 0.033), 50% OLC culture medium of OLCs cultured on bone wafer (1.128 +/- 0.028) in comparison with the pure OB group (1.393 +/- 0.016, all P < 0.05). The increase functions of OBs by OLC cultured on bone wafer and their nucleus and mitochondria were all more significant than those of the OLCs not cultured on bone wafer. The ALP activity was increased in the NaF (1.027 +/- 0.024), OCL cytoplasm (1.850 +/- 0.033), 50% OLC medium (2.074 +/- 0.065), 50% OLC medium of OLCs cultured on bone wafer (1.718 +/- 0.048), and mitochondria and cytoplasm of the OLC cultured on bone wafer groups (1.246 +/- 0.037, all P < 0.05). NaF (0.0825 +/- 0.0025), OLCs (0.0775 +/- 0.0025), nucleus (0.0775 +/- 0.0025), mitochondria (0.0875 +/- 0.0025), and cytoplasm of OLCs (0.1100 +/- 0.0007), 50% OLC medium (0.0900 +/- 0.0000), 50% OLC medium of OLCs cultured on bone wafer (0.1200 +/- 0.0041), OLCs cultured on bone wafer and nucleus, mitochondria, and cytoplasm of OLCs cultured on bone wafer all significantly increase the oeteocalcin activity of OBs (0.525 +/- 0.0063, all P < 0.05). NaF (57.6% +/- 2.6%), OLC cytoplasm (45.3% +/- 4.7%), 50% OLC medium (46.6% +/- 3.3%), 50% medium of OLCs cultured on bone wafer (54.0% +/- 2.1%), OLCs cultured on bone wafer (44.8% +/- 3.0%), and cytoplasm of OLCs cultured on bone wafer (48.7% +/- 3.5%) all significantly increased the Cbfalpha1 protein in the OBs (32.8% +/- 4.5%, all P < 0.05).
The sub-cellular elements of OLC and the supernatant of OLC culture media free of OLC promote the functions of OB, especially the OLCs cultured on bone wafer.
研究破骨样细胞（OLC）及其亚细胞结构对成骨细胞（OB）分化及功能的影响。
用5-氟尿嘧啶处理C57小鼠的脾细胞，再用白细胞介素-3、6和粒细胞-巨噬细胞集落刺激因子（GM-CSF）以及1α,25-(OH)₂D₃诱导，以获得大量OLC。将这些OLC细胞在培养液中以及在骨片上培养（称为bolcs）。培养成骨细胞，并加入氟化钠、两种OLC、不含OLC的培养液以及OLC的亚单位结构如细胞核、线粒体和细胞质，共培养5天。用MTT法测定OB的增殖率，用对硝基苯磷酸酯（PNPP）法测定碱性磷酸酶（ALP）活性。用免疫化学法检测OB中的核心结合因子α1（Cbfα1），用酶联免疫吸附测定法测定骨钙素。
与纯OB组（1.393±0.016）相比，OLC组（1.288±0.039）、OLC细胞质组（1.138±0.024）、50%OLC培养液组（1.203±0.033）、在骨片上培养的OLC的50%OLC培养基组（1.128±0.028）中的OB数量均较低（均P<0.05）。在骨片上培养的OLC及其细胞核和线粒体对OB的促进功能均比未在骨片上培养的OLC更显著。氟化钠组（1.027±0.024）、OCL细胞质组（1.850±0.033）、50%OLC培养基组（2.074±0.065）、在骨片上培养的OLC的50%OLC培养基组（1.718±0.048）以及在骨片上培养的OLC的线粒体和细胞质组（1.246±0.037）的ALP活性均增加（均P<0.05））。氟化钠组（0.0825±0.0025）、OLC组（0.0775±0.0025）、细胞核组（0.0775±0.0025）、线粒体组（0.0875±0.0025）、OLC细胞质组（0.1100±0.0007）、50%OLC培养基组（0.0900±0.0000）、在骨片上培养的OLC的50%OLC培养基组（0.1200±0.0041）、在骨片上培养的OLC以及在骨片上培养的OLC的细胞核、线粒体和细胞质均显著增加OB的骨钙素活性（0.525±0.0063，均P<0.05）。氟化钠组（57.6%±2.6%）、OLC细胞质组（45.3%±4.7%）、50%OLC培养基组（46.6%±3.3%）、在骨片上培养的OLC的50%培养基组（54.0%±2.1%）、在骨片上培养的OLC组（44.8%±3.0%）以及在骨片上培养的OLC的细胞质组（48.7%±3.5%）均显著增加OB中的Cbfα1蛋白（32.8%±4.5%，均P<0.05）。
OLC的亚细胞成分以及不含OLC的OLC培养基上清液可促进OB的功能，尤其是在骨片上培养的OLC。
