兔下颌骨来源骨髓间充质干细胞的体外培养及鉴定

The isolation and identification of bone marrow mesenchymal stem cells derived from rabbit mandible

目的:探讨兔下颌骨来源的骨髓间充质干细胞(BMMSCs)体外分离、培养并对下颌骨来源的BMMSCs进行鉴定,为骨组织工程提供种子细胞。方法:取兔下颌骨中的骨松质,采用密度梯度离心法分离单个核细胞,贴壁培养,取P2或P3代BMMSCs进行检测。倒置显微镜下观察细胞形态;MTT法测定细胞增殖并绘制细胞生长曲线;克隆形成实验计算克隆形成率;向成骨细胞、脂肪细胞、骨骼肌细胞多向诱导分化;流式细胞仪检测细胞表面抗原标记。采用SPSS16.0软件包对数据进行统计学分析。结果:细胞经传代后形态一致,呈梭形或三角形;细胞生长曲线显示,下颌骨BMMSCs经历潜伏期、对数生长期和平台期;克隆形成率为37%;成骨及成脂诱导形成钙结节及脂滴,茜素红及油红O染色呈阳性;成骨骼肌诱导desmin抗原标记阳性;流式细胞仪检测结果显示,所获得的下颌骨BMMSCs纯度较高,CD90和CD146表面标记阳性率分别为98.7%、98.1%。结论:从兔下颌骨分离的BMMSCs纯度较高,有强大的自我复制、增殖特性和体外多向诱导分化潜能,有望成为骨组织工程的种子细胞。

PURPOSE： To investigate the methods of isolation, culture and identification of BMMSCs derived from rabbit mandible. METHODS： BMMSCs were collected from rabbit mandible and isolated by density gradient centrifugation. Cells were adherently cultured in vitro, and P2 or P3 BMMSCs populations were collected and examined. Cell growth was observed by inverted microscopy; the propagation of BMMSCs were tested by MTF and a growth curve was drawn after statistical analysis; colony-forming unit-fibroblast （CFU-F） was detected by examination of colony formation; the potential of multi-directional differentiation into osteoblasts, adipocytes and skeletal muscle cells was estimated by pertinent methods; the surface marks of BMMSCs were detected by flow cytometry;the data was analysed using SPSS16.0 software package. RESULTS： The majority of adherent cells were long fusiform, and few were small triangle; the growth curve of BMMSCs showed that every passage experienced incubation period, log phase and platform period; the rate of colony formation was 37%. Growth of BMMSCs represented the appearance of CFU-F; BMMSCs after inducted differentiation showed osteogenic and adipogenic potential. The staining of mineralized nodules was positive by alizarin red S and the positive staining of oil red O appeared in lipid drops around cell nucleus. The staining of skeletal muscle cells was positive by desmin immunofluorescence; the cell surface marks assessed with flow cytometry indicated that these BMMSCs expressed CDg0 and CD146 in high percentage （about 98.7% and 98.1%, respectively）. CONCLUSIONS： The highly uniformed BMMSCs derived from rabbit mandible can be collected. These BMMSCs have the ability of self-replication and propagation, as well as potential of multi-directional differentiation in vitro. Supported by National Natural ScienceFoundation of China（81170935）.