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目的:研究体外应用不同培养方法对所生成的破骨细胞数量及功能的影响。方法:体外采用3种方法培养破骨细胞:A组小鼠骨髓细胞中加入10 ng/ml巨噬细胞集落刺激因子(M-CSF)培养24 h,未贴壁细胞30 ng/ml M-CSF预诱导3 d后,50ng/ml M-CSF+100 ng/ml核因子κB受体活化因子配体(RANKL)继续诱导;B组小鼠骨髓细胞与小鼠颅骨成骨细胞以10∶1的比例混合培养,加入1×10-6mol/L前列腺素E2(PGE2)和1×10-8mol/L维生素D3(VitD3);C组小鼠单核巨噬细胞系RAW264.7中加入100 ng/ml RANKL诱导培养。检测每组细胞抗酒石酸酸性磷酸酶(TRAP)染色情况及牙本质磨片吸收陷窝情况,Real-time PCR检测各组破骨细胞NFATc1、c-Fos表达情况。结果:各组细胞均有TRAP阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝。B组所形成的破骨细胞数量最多,C组次之,A组最少;B组骨吸收陷窝数目最多,陷窝总面积最大,A组其次,C组最差;B组NFATc1、c-Fos表达高于C组及A组,A组表达最差。结论:3种培养破骨细胞的方法相比较,B组在破骨细胞分化和吸收功能方面优于A、C组。A、C组相比较,A组培养的破骨细胞骨吸收功能更强,C组所培养的破骨细胞分化更佳。
Objective: To study the effects of different culture methods on the number and function of osteoclasts produced in vitro. Methods: Three kinds of osteoclasts were cultured in vitro. Group A bone marrow cells were cultured with 10 ng / ml macrophage colony-stimulating factor (M-CSF) for 24 h, and non-adherent cells 30 ng / ml M-CSF After 3 days of pre-induction, 50 ng / ml M-CSF + 100 ng / ml RANKL was continued to induce; Group B mouse bone marrow cells and mouse calvarial osteoblasts were treated with 10: 1 1 × 10-6mol / L prostaglandin E2 (PGE2) and 1 × 10-8mol / L vitamin D3 (VitD3) were added into the mixed culture of RAW264.7 cells. ml RANKL induction culture. The tartrate-resistant acid phosphatase (TRAP) staining of each group and the absorption lacunae of dentinal abrasives were detected. The expression of NFATc1 and c-Fos in osteoclasts of each group was detected by Real-time PCR. Results: TRAP-positive multinucleated osteoclasts were all formed in each group of cells, and the absorption lacuna was formed on the dentine disk. The number of osteoclasts formed in group B was the highest, that in group C was second, and that in group A was the lowest. The number of bone resorption lacunae in group B was the highest, the total area of lacuna was the largest in group B, Fos expression was higher than that in group C and group A, and group A had the worst expression. CONCLUSION: Compared with the methods of culturing osteoclasts, B group is superior to A and C groups in osteoclast differentiation and absorption function. Compared with group A and group C, osteoclasts cultured in group A were more effective in bone resorption and osteoclasts cultured in group C were better differentiated.