大鼠骨髓单核细胞的快速分离方法及向破骨细胞的诱导分化

A method about isolation and cultivation of rat bone marrow monocytes and differentiation into osteoclasts

摘要:

目的 建立一种简便高效的体外分离大鼠骨髓单核细胞的方法，观察其体外生长特性，并诱导其分化为破骨细胞。方法无菌条件下分离出SD大鼠的胫骨和股骨，使用环氧树脂管（EP 管）和移液枪头快速分离骨髓组织，再用红细胞裂解液去除红细胞。细胞培养过夜后收集悬浮细胞，加入巨噬细胞集落刺激因子（M-CSF）继续培养扩增后得到贴壁的骨髓单核细胞。观察骨髓单核细胞生长过程中的形态学特征；通过细胞计数试剂盒法（CCK-8）测绘细胞的生长曲线；用流式细胞仪检测细胞表面CD11b 的表达；在加入M-CSF 和核因子κB受体活化因子配基（RANKL）诱导分化后，用抗酒石酸酸性磷酸酶（TRAP）染色和降钙素受体（CTR）免疫荧光染色鉴定其是否能分化为成熟破骨细胞。结果通过该方法获得的大鼠骨髓单核细胞培养1 d后基本呈小圆形，杂细胞较少。3 d后细胞数目稍多，两端开始出现触角，5 d后数目增多，细胞呈椭圆形，两端触角明显；细胞的增殖依赖于M-CSF；流式结果显示，通过此方法获得的单核细胞纯度较高；TRAP 染色和CTR 免疫荧光染色结果提示，通过该方法获得的单核细胞可以诱导为成熟破骨细胞。结论通过EP管和移液枪头装置可快速分离 获取单核细胞，获得的细胞表型稳定，适合用于骨代谢疾病的进一步研究。

Abstract:

Objective To establish a simple and efficient method to isolate the rat bone marrow monocytes in vitro, observe the characteristics of growth and induce the differentiation of osteoclasts. Methods The tibia and femur of SD rats were isolated under aseptic condition. The bone marrow tissues were separated by EP tube and the transfer gun head. Red blood cell lysate was used to remove the red blood cells. The suspended cells were collected after cell culture overnight. Then M-CSF was added to culture the cells and adherent bone marrow monocytes were obtained. The growth process of bone marrow monocytes was morphologically observed. The cell growth curve was measured by CCK -8. Flow cytometry was used to detect CD11b expression on cell surface. After differentiation of the monocytes by adding M-CSF and RANKL, TRAP staining and CTR immunofluorescence staining were used to identify whether they can differentiate to mature osteoclasts. Results The rat bone marrow monocytes obtained after culturing for 1 day by these methods showed small circular shape with few mixed cells. After 3 days, the number of the cells increased slightly and the antennae began to appear at both sides. After 5 days, the cells increased significantly and were in oval shape, the antennae at both sides were obvious. The cell proliferation depended on M-CSF. Flow cytometry showed that monocytes obtained had high purity by this method. TRAP staining and CTR immunofluorescence staining results showed that monocytes obtained by this method could be successfully induced to mature osteoclasts. Conclusions Monocytes can be isolated and obtained rapidly by this method. The phenotype of the obtained cells is stable and suitable for further study on bone metabolic diseases.