不同培养方法骨髓破骨细胞样细胞分化及活性的实验观察

An experimental study of the differentiation and function of osteoclast-like cells derived from bone marrow cells by different cultural methods

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 compare the differentiation and function of osteoclast-like cells（OLC） derived from bone marrow cells by different cultural methods. Methods Rat bone marrow cells were incubated with α-MEM containing macmphage colony stimulating factor （M-CSF） for two hours, then cultured with α- MEM containing M-CSF and receptor activator of NF-κB ligand （RANKL） for 7 days. The culture was divided into two groups：group A, whole bone marrow cells were cultured; group B, bone marrow cells were cultured after density gradient centrifugation. The differentiation of OLC was studied by morphologic features and TRAP staining, while the function difference of OLC was studied by analyzing of bone absorptive lacunae and integrin 133 （CD61）. Result The amount of TRAP-positive multinucleated OLCs and bone absorptive lacunae, the activity of OLC TRAP in group B were higher than group A at day 5 and day 7 （P〈0.01, P〈0.05）. The area ratio of bone absorptive lacunae had no difference in cultural nonage between two groups but had obviously distinction at day 7; The expression of CD61 were higher in cultural nonage, but had no statistical difference in cultural anaphase. Conclusion Low rate of rotation and spurt density gradient centrifugation has no feffect on the differentiation and function of OLC derived from bone marrow cells. This cultural method was convenient and potent.