小鼠骨髓间充质干细胞分离方法的比较与探讨  

Comparison and discussion of methods for isolating mouse bone marrow mesenchymal stem cells

作　　者：廖新爱 蔡丹妮 游若兰 黄慧芳[1] LIAO Xinai;CAI Danni;YOU Ruolan;HUANG Huifang(Central Laboratory of Fujian Medical University Union Hospital,Fujian University,Fuzhou 350001,China)

机构地区：[1]福建医科大学附属协和医院中心实验室,福州350001

出　　处：《中国实验动物学报》2023年第1期1-9,共9页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(82070145);高水平实验研究平台建设项目(闽201704)。

摘　　要：目的探讨可高效分离小鼠骨髓间充质干细胞(mBMSCs)的技术方法。方法分别采用骨片消化爬片法、全骨髓贴壁法、骨片消化液上清法和骨片消化研钵法等四种方法分离7~9周龄雄性C57BL/6小鼠的mBMSCs;于倒置显微镜下观察原代mBMSCs形态;运用流式细胞术检测mBMSCs特征性免疫表型;采用多向分化诱导检测mBMSCs成骨分化能力与成脂分化能力。结果分离获得的原代细胞首次换液后于倒置显微镜下观察:骨片消化爬片法分离的细胞,仅见大量骨碎片和杂细胞,该法所分离细胞不再进行后续检测评估;全骨髓贴壁法分离的细胞,仅可见少量多角形贴壁细胞,夹杂大量杂细胞;骨片消化液上清法分离的细胞也可见较多长梭形、三角形贴壁细胞,但杂细胞较多;骨片消化研钵法分离的细胞,可见较多长梭形、多角形贴壁细胞,杂细胞少。分离细胞经培养传代到第3代(P3代)时,一方面采用流式细胞术分析mBMSCs特征性免疫表型,结果表明骨片消化液上清法和骨片消化研钵法分离的mBMSCs纯度均较高,全骨髓贴壁法不理想;另一方面,进行成骨分化诱导与成脂分化诱导,结果表明与全骨髓贴壁法分离的细胞相比,骨片消化液上清法和骨片消化研钵法所分离出的细胞具有较强的多向分化潜能。结论骨片消化液上清法和骨片消化研钵法分离获得的mBMSCs纯度较高、质量较好。Objective To achieve the efficient isolation of mouse bone marrow mesenchymal stem cells(mBMSCs).Methods mBMSCs from C57BL/6 mice,aged 7 to 9 weeks,were isolated by four method:the bone fragment digestion and crawl method,whole⁃bone⁃marrow adherent method,bone fragment digestive fluid supernatant method,and bone fragment digestion and mortar method.An inverted microscope was used to observe the morphology of the mBMSCs,and their characteristic immunophenotype was detected by flow cytometry.The osteogenic and adipogenic differentiation abilities of mBMSCs were detected through multidirectional differentiation induction.Results Isolated primary cells were observed under an inverted microscope after the first fluid change.A large number of bone fragments and miscellaneous cells only were found in the primary cells isolated by the bone fragment digestion and crawl method,and this method was no longer evaluated.Only a small number of polygonal adherent cells and a large number of miscellaneous cells were observed in the whole⁃bone⁃marrow adherent⁃extracted sample.The cells isolated by the bone fragment digestive fluid supernatant method were longer spindle⁃shaped and triangular adherent cells,but there were many miscellaneous cells.The cells isolated by the bone fragment digestion and mortar method were longer spindle⁃shaped and polygonal adherent cells,and there were fewer miscellaneous cells.The isolated cells were cultured to passage 3,and the characteristic immunophenotypes of mBMSCs were analyzed by flow cytometry.The result showed that the purity of mBMSCs isolated via the bone fragment digestive fluid supernatant method and bone fragment digestion and mortar method was higher than that of cells isolated via the whole⁃bone⁃marrow adherent method.When osteogenic differentiation and adipogenic differentiation were induced,cells isolated by the bone fragment digestive fluid supernatant method and bone fragment digestion and mortar method had stronger multidirectional differentiation potential than

关 键 词：小鼠骨髓间充质干细胞 骨片消化爬片法 全骨髓贴壁法 骨片消化液上清法 骨片消化研钵法 

分 类 号：Q95-33[生物学—动物学]