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目的研究不同骨髓细胞培养方法对大鼠破骨细胞样细胞(OLC)诱导分化及活性的影响。方法大鼠骨髓细胞以巨噬细胞集落刺激因子(M-CSF)作用后与核因子!B受体活化因子配体(RANKL)协同诱导OLC分化,采用全骨髓细胞(A组)及密度梯度离心后骨髓单个核细胞(B组)两种培养方法,通过倒置相差显微镜及细胞染色观察两组OLC分化数量的差异,通过骨吸收陷窝分析及OLC膜表面整合素#3(CD61)表达量比较两组OLC活性的差异。结果B组培养5、7d抗酒石酸酸性磷酸酶(TRAP)染色阳性多核OLC数量及TRAP活性均高于A组,骨吸收陷窝计数亦较A组为多,差异均有统计学意义(P<0.01,P<0.05);陷窝面积比培养早期两组比较差异无统计学意义,7d时B组大于A组,差异有统计学意义(P<0.01);OLC膜表面CD61表达量及平均荧光强度0及3d时B组均较A组明显增高,随培养时间延长组间比较差异无统计学意义。结论低转速、短时间密度梯度离心后骨髓细胞培养诱导OLC分化获得的细胞数量较多且不影响其活性,是一种简便有效的OLC培养方法。
Objective To study the effects of different methods of bone marrow cell culture on the differentiation and activity of rat osteoclast-like cells (OLCs). Methods Rat bone marrow cells were induced to act as macrophages colony-stimulating factor (M-CSF) and combined with RANKL to induce OLC differentiation. Whole bone marrow cells (A group) and density gradient centrifugation (B group). The difference of OLC differentiation between the two groups was observed by inverted phase contrast microscope and cell staining. Through the bone resorption lacuna analysis and the expression of integrin # 3 (CD61) on the surface of OLC Differences in OLC activity between the two groups. Results The number of TRAP - positive multinuclear OLC and the activity of TRAP in group B were higher than that in group A at 5 and 7 days after transplantation. The number of bone resorption lacuna was also higher in group B than in group A (P < 0.01, P <0.05). There was no significant difference in the area of lacuna between the two groups at the early stage of culture, and the level of CD61 on the surface of OLC was significantly higher than that of group A on the 7th day (P <0.01) At 0 and 3 days, the intensity of B group was significantly higher than that of A group, and there was no significant difference between the two groups as the training time prolonged. Conclusion It is a simple and effective OLC culture method that the number of cells induced by OLC differentiation after low-speed and short-term density gradient centrifugation is large and does not affect its activity.