目的通过将鼠成骨细胞与破骨细胞直接共培养的实验方法,研究成骨细胞对破骨细胞分化及功能成熟的影响。方法按照M-CSF 30μg/L、RANKL 50μg/L的浓度对鼠骨髓单个核细胞诱导培养6 d,与原代培养3 d的鼠成骨细胞按细胞数量1∶1直接共培养,加入1,25-(OH)2D31×10^-8mol/L和PGE21×10^-6mol/L,利用形态学观察、TRAP染色、骨吸收陷窝检测等方法对共培养细胞进行鉴定。结果成骨细胞与破骨细胞共培养时,成骨细胞有明显的生长优势;染色后显微镜下可见大量呈单层排列的成骨细胞,偶见TRAP(+)的破骨细胞。结论成骨细胞对破骨细胞分化及功能成熟的影响与两者的相对数量有关。本实验中,由于成骨细胞快速生长,当其数量多于破骨细胞时,可能使1,25-(OH)2D3对成骨细胞RANKL表达的上调作用不足,且PGE2对成骨细胞OPG表达的下调作用不足,从而主要表现为对破骨细胞形成及分化的抑制作用。
Objective By culturing the osteoclasts together with the osteoblasts directly to investigate the effect of osteoblasts on the formation of mature osteoclasts. Methods The bone marrow mononuclear cells of rats were treated with 30 μg/L M-CSF and 50 μg/L RANKL and cultured for 6 days. Subsequently, the primary osteoblasts which were of the same quantity as the osteoclasts were co-cultured directly. In the co-culture system, we added the liquid containing 1, 25-(OH)2D3 1 × 10^-8 mol/L and PGE2 1 × 10^-6 mol/L. The morphological observation, TRAP staining and pit staining were adopted to identify osteoclasts. Results When the osteoclasts were co-cultured with primary osteoblasts, the growth of osteoblasts had more preponderances. After staining, we could see more osteoblasts than osteoclasts. Oonciusion The relationship between osteoblasts and osteoclasts is related to the relative quantities of the two cells. When osteoblasts outnumber osteoclasts, osteoblasts would inhibit the formation and differentiation of osteoclasts.