人骨髓间充质干细胞分离鉴定方法的改进

A modified Method to Isolate and Identify the Adult Mesenchymal Stem Cells from Human Bone Marrow

本研究的目的是建立一种基于临床移植需要的分离、培养、鉴定人骨髓间充质干细胞(mesenchymalstemcells,MSC)的方法,为配合造血干细胞移植的临床应用提供实验基础。采用Percoll分离液(1.073g/ml)从新鲜成人骨髓穿刺液中分离MSC,应用贴壁筛选法传代培养,流式细胞仪检测细胞表面抗原,成骨诱导体系(地塞米松0.1μmol/L、β-甘油磷酸钠10mmol/L、抗坏血酸磷酸盐50μmol/L)及脂肪诱导体系(地塞米松1μmol/L、牛胰岛素5mg/L、1-甲基-3-异丁基-黄嘌呤0.5mmol/L、消炎痛60μmol/L)分别诱导MSC定向分化,通过细胞形态观察及免疫化学染色进行鉴定。结果表明:从成人骨髓液中分离培养出MSC,稳定表达CD73、CD105、CD166,不表达CD34、CD45。定向诱导的成骨细胞表达碱性磷酸酶活性,脂肪细胞内出现明显的脂滴。结论:采用密度梯度离心联合贴壁筛选法并通过流式细胞术检测及定向诱导分化,能够从成人骨髓液中分离出MSC,并完成细胞表型及多向分化潜能的初步鉴定。本操作方案具有一定的临床应用价值。

The study was aimed to establish a protocol of isolating and culturing adult mesenchymal stem cells （MSC） from human bone marrow aspirate and identify them by surface antigen analysis and committed differentiation in order to provide an experimental foundation for achieving a therapeutic benefit in applying MSC in hematopoiefic stem cell transplantation. MSCs were obtained from flesh human bone marrow aspirate by gradient centrifugation with Percoll （1.073 g/ml） and anchoring culture in L-DMEM with 10% fetal bovine serum by a full medium exchange every 3 days. The MSC surface antigens, including CD34, CD45, CD73, CD105, CD166, were analyzed on FACScan flow cytometer, Under culture in conditioned medium for osteogenesis （ the hormone cocktail containing 0, 1 μmol/L dexamethasone, 10 mmol/L glycerol-2-phosphate and 50 μmol/L ascorbic acid） and adipogenesis （ the cocktail containing 1 μmol/L dexamethasone, 5 mg/L insulin, 0.5 mmool/L 1-methyl-3-isobutylxanthine and 60 μmol/L indomethacin）, MSCs cornmittedly differentiated into osteoblasts and adipocytes, The differentiated mesenchymal stem cells were identified by morphological observation and immunohistochemical staining, The results showed that by gradient centrifugation and adhesion culture, MSCs could be isolated and culture-expanded from human bone marrow aspirate, These cells were uniformly negative for CD34, CD45 and positive for CD73, CD105 and CD166. The osteogenic differentiated cells were positive for alkaline phosphatase （ALP） and the adipogenic differentiated cells displayed accumulation of lipid vacuoles, as detected by oil red O, It is concluded that MSC can be isolated and expand-cultured from adult human bone marrow aspirate and committedly differentiate into osteoblasts and adipocytes. MSC primary identification can be accomplished by flow cytometry and induced differentiation, The set of methods in current experiment shows somewhat practical value for basic research and clinical application.