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

发布时间：2018-05-19 11:58

本文选题：分离培养方法 + 破骨细胞　；参考：《中国现代医学杂志》2017年18期

【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 for isolating rat bone marrow monocytes in vitro and to observe their growth characteristics in vitro and induce them to differentiate into osteoclasts. Methods the tibia and femur of SD rats were isolated under aseptic condition. The bone marrow tissue was separated quickly by using epoxy resin tube and EP tube. Then the erythrocyte was removed by erythrocyte lysis solution. Suspension cells were collected after overnight cell culture, and macrophage colony stimulating factor (M-CSF) was added to obtain adherent bone marrow monocytes. Morphological characteristics of bone marrow monocytes were observed, cell growth curve was measured by cell count kit (CCK-8), CD11b expression on cell surface was detected by flow cytometry. After induction of differentiation by adding M-CSF and nuclear factor- 魏 B receptor activator ligand (RANKL), the differentiation of osteoclasts was identified by tartrate-resistant acid phosphatase staining and calcitonin receptor immunofluorescence staining. Results the monocytes of rat bone marrow obtained by this method were basically small round after 1 day culture, the number of cells was slightly more after less than 3 days, the number of cells increased after 5 days, the number of cells was oval, and the antennae of both ends were obvious. The results of flow cytometry showed that the monocytes obtained by this method were highly purified by trap staining and CTR immunofluorescence staining, which indicated that the monocytes obtained by this method could be induced into mature osteoclasts. Conclusion Mononuclear cells can be rapidly isolated and obtained by EP tube and liquid transfer device. The obtained cells have stable phenotypes and are suitable for further study of bone metabolic diseases.
【作者单位】：南京中医药大学骨伤研究所(骨伤修复与重建新技术实验室);南京中医药大学附属医院骨伤科;
【基金】：国家自然科学基金面上项目(No:81473692) 江苏省自然科学基金青年基金项目(No:BK20151007)
【分类号】：R329.2

【相似文献】