体外应用不同方法培养破骨细胞的实验对比研究
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国家自然科学基金(81270965);河北联合大学科学研究基金(z201233)


The different methods of osteoclast culture in vitro
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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,50 ng/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 × 10-6 mol/L 前列腺素E2(PGE2)和1 × 10-8 mol/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。

    Abstract:

    Objective:To study the influence of osteoclast generation and function in different methods of osteoclast culture in vitro. Methods:We used 3 methods of osteoclast culture in vitro:the group A,10 ng/ml M-CSF was added in mice marrow cell for 24 h. Cells which not adhere to the surface were pre-induction with 30 ng/ml M-CSF for 3 d,and then 50 ng/ml M-CSF + 100 ng/ml RANKL were added;the group B,mix culture mice marrow cell with mice osteoblast on 10∶1,and 10-6 mol/L PGE2 and 10-8 mol/L VitD3 were added;the group C,RAW264.7 was added with 100 ng/ml RANKL to inducement. Osteoclastogenesis and their resorption function were examined by TRAP staining and scanning electron microscope (SEM) observation of dentin resorption lacunaes. The gene expressions of NFATc1 and c-Fos were also detected by Real-time PCR. Results:TRAP positive multinuclear cells were observed and resorption lacunaes were formed in each group. However,the group B showed more TRAP positive multinuclear cells and larger amount of resorption lacunaes than the group A and C. The group C showed more TRAP positive multinuclear cells but less amount of resorption lacunaes than the group A. Real-time PCR detection also showed that the gene expressions of NFATc1 and c-Fos were higher in the group B than the group A and C. Conclusion:As the comparison of three methods of osteoclast culture,the group B is better than the group A and C in osteoclastogenesis and resorption function. The group A is better than the group C in the resorption function of osteoclast,but the group C is better than the group A in osteoclastogenesis.

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董 伟,冯晓洁,戚孟春,梁永强,彭宏峰.体外应用不同方法培养破骨细胞的实验对比研究[J].南京医科大学学报(自然科学版),2013,(4):475-479

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  • 收稿日期:2012-11-04
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  • 在线发布日期: 2013-04-29
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