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目的:研究蛇床子总香豆素(TCFC)对破骨细胞的作用,探讨其抗骨质疏松的作用机制。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D_3和地塞米松作用下,使骨髓单核细胞分化形成破骨细胞。通过相差显微镜下的形态观察,抗酒石酸酸性磷酸酶染色和骨片上骨吸收陷窝的形成来鉴定破骨细胞。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,原子吸收光谱法测定破骨细胞和骨片联合培养的培养上清中钙的浓度。结果:蛇床子总香豆素(TCFC)2.5—25mg/L抑制破骨细胞的形成和分化。TCFC0.25-25mg/L作用24-72h,可以显著抑制抗酒石酸酸性磷酸酶的活性,TCFC 25mg/L作用48h和72h分别可使其降低26.3%和24.1%。TCFC 25mg/L可使破骨细胞在骨片上形成的吸收陷窝的面积减少25.05%,骨片中Ca~(2+)的释放减少41.73%。结论:TCFC通过抑制破骨细胞的形成、抗酒石酸酸性磷酸酶的活性和骨吸收作用减少骨质丢失。
Objective: To study the effect of total coumarin (TCFC) on osteoclasts and explore its anti-osteoporosis mechanism. METHODS: Primary cultured osteoblasts and bone marrow mononuclear cells were co-cultured to induce bone marrow mononuclear cells to differentiate into osteoclasts under the action of 1,25-(OH)2 vitamin D3 and dexamethasone. Osteoclasts were identified by morphological observation under phase contrast microscopy, tartrate acid phosphatase staining and the formation of bone resorption lacunae on bone fragments. The activity of tartrate-resistant acid phosphatase in osteoclasts was determined by disodium phosphate method. The area of resorption lacunae on bone fragments was measured by computer image analysis technique. The culture of osteoclasts and bone chips was determined by atomic absorption spectrometry. Clear calcium concentration. Results: The total coumarin (TCFC) 2.5-25 mg/L inhibited the formation and differentiation of osteoclasts. Treatment with TCFC0.25-25mg/L for 24-72h could significantly inhibit the activity of tartrate-resistant acid phosphatase. TCFC 25mg/L for 48h and 72h could reduce them by 26.3% and 24.1%, respectively. TCFC 25mg/L can reduce the area of the absorption lacunae formed on osteoblasts by 25.05%, and the release of Ca~(2+) in bone fragments decreases by 41.73%. CONCLUSION: TCFC reduces bone loss by inhibiting the formation of osteoclasts, the activity of tartrate-resistant acid phosphatase, and bone resorption.