背景: 传统治疗肌组织缺损的方法是肌肉转位替代疗法,这样就造成了另一块肌组织的损伤.在这种背景下,作者设计了该课题,寻找肌肉原位修复的方法.目的: 探讨大鼠骨髓间充质干细胞在体外诱导分化为骨骼肌细胞的条件.方法: 分离培养大鼠骨髓间充质干细胞,取第3代骨髓间充质干细胞,应用5一氮杂胞苷、成肌分化因子、转化生长因子β1、胰岛索样生长因子1联合进行诱导,使之分化.在诱导后的第9天,收集细胞,免疫组织化学法对细胞进行鉴定.结果与结论: 原代培养的骨髓间充质干细胞呈贴壁、集落样生长,5～7 d后有多突成纤维细胞、扁平多角形细胞、多角形细胞和三角形细胞.12 d后,见细胞融合,铺满瓶底,骨髓间充质干细胞的形态变化不明显.经5-氮杂胞苷诱导后细胞,起初部分细胞死亡,生长缓慢.7 d后见细胞明显生长,体积逐渐增大,呈椭圆形、梭形或不规则形状.14 d后,大量增殖的长梭形细胞开始增多.18～22 d后,肌管数量增多,体积增大,核数亦明显增多,初生肌管与长梭形成肌细胞呈平行排列,间隔分布.骨髓间充质/干细胞免疫组化法测定见CD44反应阳性,胞质呈棕褐颗粒,核周明显,CD34呈阴性反应.诱导后骨髓间充质干细胞免疫组化法测定见结蛋白、骨骼肌特异肌球蛋白均为阳性表达.结果提示在体外,成肌分化因子和5-氮杂胞苷可以诱导骨髓间充质干细胞向骨骼肌细胞定向分化,并伴有结蛋白和骨骼肌肌球蛋白阳性表达.%BACKGROUND:Muscle transposition is a conventional method to treat muscle tissue defects,but it results in damage to another piece of muscle.For this reason,we designed this study to search for a method to in situ repair muscle tissue defects.OBJECTIVE:To investigate the conditions for in vitro induced differentiation of rat bone marrow mesenchymal stem calls(BMSCs)into skeletal muscle cells.METHODS:Following isolation and culture,passage 3 BMSCs were induced to differentiate in vitro by a combination of5-azacytidine,myogenic differentiation factor,transforming growth factor β1,and insulin like growth factor.At 9 days after induction,cells were harvested and identified by immunohistochemistry.RESULTS AND CONCLUSION:Primary cultured BMSCs exhibited an adherent,colony-like growth.After 5-7 days,multi-synaptic calls,thin and fiat polygonal cells,polygonal cells,and triangle-shaped cells were observed.After 12 days,calls confluenced and covered the whole bottom of culture flask,with slightly altered morphology of BMSCs.After 5-azacytidine induction,some calls died and grew slowly.After 7 days,cells markedly grew and soma was gradually enlarged,presenting with an oval,spindle-shaped,or irregular appearance.After 14 days,spindle-shaped calls become more.After 18-22 days,myotubes were increased in number and enlarged in volume,and myotube nucleuses were also increased.The newly formed myotubes and spindle-shaped fibroblasts were distributed in parallel interval.The immunohistochemistry of BMSCs revealed that cells were positive for CD44,with dark brown granules in the cytoplasm,especially around the nucleus,but they were negative for CD34.The immunohistochemistry of induced BMSCs demonstrated that calls were positive for desmin and skeletal muscle myosins.These findings indicate that myogenic differentiation factors and 5-azacytidine could induce the oriented differentiation of BMSCs into skeletal cells,with the presence of positive expression of desmin and skeletal muscle myosins.
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