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目的:研究小檗碱对破骨细胞分化及骨吸收功能的影响,探讨小檗碱抑制骨吸收的细胞学基础。方法:采用原代培养的成骨细胞和骨髓单核细胞联合培养的方法,在1,25-(OH)2维生素D3和地塞米松作用下,使骨髓单核细胞分化形成破骨细胞。通过相差显微镜观察细胞形态,通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色和观察骨片上骨吸收陷窝的形成鉴定破骨细胞。磷酸苯二钠法测定破骨细胞抗酒石酸酸性磷酸酶的活性,计算机图像分析技术测定骨片上破骨性骨吸收陷窝的面积。结果:小檗碱在0.1～10μmol/L范围内,浓度依赖性地抑制TRAP阳性多核破骨细胞的形成和TRAP活性,减少破骨性骨吸收陷窝的面积;在10μmol/L浓度下,对破骨细胞的抑制作用最强,对TRAP阳性多核破骨细胞的形成和TRAP活性的抑制率分别达到了60.45%和42.12%,骨吸收陷窝面积减少72.69%。结论:小檗碱通过抑制破骨细胞的形成、分化和骨吸收功能减少骨质的丢失。
Objective: To study the effect of berberine on osteoclast differentiation and bone resorption, and to explore the cytological basis of berberine inhibiting bone resorption. Methods: Primary culture of osteoblasts and bone marrow mononuclear cells co-culture method, 1,25- (OH) 2 vitamin D3 and dexamethasone under the action of bone marrow mononuclear cells to form osteoclasts. Cell morphology was observed by phase contrast microscopy, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRAP) staining and bone resorption lacuna formation on the bone chip. The activity of tartrate-resistant tartrate-resistant acid phosphatase was determined by phenyl-disodium phosphate method, and the area of osteoclastic bone resorption lacuna was measured by computer image analysis. Results: Berberine inhibited the formation of TRAP-positive multinucleated osteoclasts and TRAP activity in a concentration-dependent manner from 0.1 μmol / L to 10 μmol / L, and decreased the area of osteoclastic bone resorption lacuna. At the concentration of 10 μmol / L, The inhibitory effect of osteoclasts was the strongest, which inhibited the formation of TRAP-positive multinucleated osteoclasts and the inhibition rate of TRAP activity respectively by 60.45% and 42.12%, and the bone resorption lacuna area decreased by 72.69%. Conclusion: Berberine can reduce the loss of bone mass by inhibiting osteoclast formation, differentiation and bone resorption.