小鼠骨骼肌源间充质干细胞的分离与培养

Development of Methodology for Isolation and Culture of Mesenchymal Stem Cells Derived from Mouse Skeletal Muscle

为观察成体小鼠肌肉组织中是否含有间充质干细胞 (mesenchymalstemcells ,MSC) ,将分离胶原酶消化的肌肉单个核细胞 ,在含 10 %胎牛血清的α MEM /F12培养液中培养 ,观察培养细胞的形态、细胞化学及增殖特性特征 ,用流式细胞术分析细胞表型 ,并观察细胞体外向成骨细胞分化能力。结果显示 ,从小鼠肌肉组织中分离和培养获得的细胞具有良好的增殖特性 ,CD3 4及CD45阴性 ,CD2 9及Sca 1阳性率在 90 %以上。细胞化学染色显示 ,细胞非特异性酯酶和酸性磷酸酶为阳性 ,碱性磷酸酶阴性 ,糖原为弱阳性。细胞经维生素C ,β 磷酸甘油及地塞米松诱导后 ,碱性磷酸酶活性明显增强 ,阳性率达到 94%以上。结论 :成体小鼠肌肉组织中含有间充质干细胞 。

To address the question whether there exists mesenchymal stem cells in adult mou se skeletal muscle, mononuclear cells from muscle were obtained by digestion and density gradient centrifugation and plated in α-MEM/F12 medium containing 10 % fetal bovine serum. Cell biological properties including morphology, cytochemist ry, growth pattern and phenotypes as well were evaluated. Likewise, the osteogen esis of cultured cells was also observed. The results showed that adherent cells homogenous in shape proliferated quickly in the culture system. The phenotypes of the cells were unique, which were positive for CD29 and Sca-1, and negat ive for CD34 and CD45. Cytochemistry evaluation showed that they were homogeneou sly positive for acid α-naphthl acetate esterase (ANAE) and acid phosphatase ( A CP), and negative for alkaline phosphatase (ALP) and that, around 5% of them wer e positive for glycogen (periodic acid-Schiff reaction, PAS). Cells became ALP -positive after the induction by ascorbic acid, β-phosphoglycerol and dexamet hasone. It is concluded that mesenchymal stem cells exist in murine skeletal mu scle and compose the complex heterogenous population of stem cells in muscle.