Quinn J M, Morfis M, Lam M H, Elliott J, Kartsogiannis V, Williams E D, Gillespie M T, Martin T J, Sexton P M
St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, The University of Melbourne, Fitzroy, Victoria, Australia.
Bone. 1999 Jul;25(1):1-8. doi: 10.1016/s8756-3282(99)00094-0.
Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.
破骨细胞是负责骨吸收的细胞，其数量和形成速率对于确定骨量至关重要。为了识别和量化破骨细胞，并研究它们在骨组织和破骨细胞生成培养物中的形成过程，需要破骨细胞特异性细胞标志物。只有降钙素受体（CTR）表达能明确识别破骨细胞，并将它们与巨噬细胞多核体区分开来。然而，目前用于CTR检测的放射自显影方法繁琐且耗时。我们已经开发出针对小鼠和大鼠Cla CTR细胞内C末端结构域的兔多克隆抗体。这些抗体标记了稳定转染CTR的HEK-293细胞（未转染的HEK-293细胞则未被标记）。用重组抗原预吸收抗体可消除这种标记。这些抗体对原代小鼠和大鼠破骨细胞以及小鼠骨切片中的破骨细胞进行了免疫染色。在1,25-二羟维生素D3和前列腺素E2存在的情况下，由小鼠骨髓或脾细胞与成骨细胞共培养形成的破骨细胞（单核和多核）也被CTR抗体特异性免疫染色。在不允许破骨细胞生成的条件下培养的共培养物（即省略介质或成骨细胞，或培养少于4天）未被CTR抗体免疫染色。125I标记的鲑鱼降钙素的放射自显影检测与CTR免疫组织化学相结合表明，两种方法标记的是相同的细胞。将CTR多克隆抗体和单克隆抗体F4/80联合使用，分别显示了骨髓/成骨细胞共培养物中鼠破骨细胞和巨噬细胞群体的免疫荧光标记。这些结果表明，基于CTR抗体的简单快速方法可用于识别破骨细胞，并可通过双免疫荧光分析用于表征破骨细胞的抗原谱。
