小鼠骨髓间充质干细胞体外诱导分化的形态学观察

Morphologic Observation on Differentiation of Mouse Mesenchymal Stem Cells Induced in Vitro

目的探讨5-氮胞苷(5-aza)对小鼠骨髓间充质干细胞(MSCs)的体外诱导作用。方法分离2周龄小鼠胫骨、股骨,冲洗出骨髓,利用Ficoll淋巴细胞分离液密度梯度离心和贴壁培养的方法获得MSCs。取传2代MSCs培养48h后加10μmol/L5-aza进行诱导,3周后刮取贴壁细胞,离心收集,免疫细胞化学检测心肌特异性蛋白α-actin的表达对所诱导细胞进行鉴定,电镜观察细胞超微结构。结果电镜下见胞质内出现大量肌丝,线粒体数量增多,分布在肌丝周围,可见细胞膜电子密度增高的缝隙连接;免疫细胞化学显示α-actin呈强阳性表达。结论MSCs经5-aza体外诱导分化,可获得形态上类似、表达心肌特异性蛋白的心肌样细胞。

Objective To investigate the inducing effect of 5-aza on mouse mesenchymal stem cells in vitro. Methods The mononuclear cells were isolated from tibias and fibulas of 2 weeks old mice and the mesenchymal stem cells of bone marrow were obtained with the combination of gradient centrifugation and cellular attaching culture method. Cells of the 2nd passage after 48 hours＇ culture were treated with 10μmol/L 5-aza for 3 weeks. Tben collect the cells and separate them into two parts,one for identification by detecting α-cardiac specific actin,the other for observation by electron microscope. Results Treated with 10μmol/L 5-aza,some cells had a clear myofilament like with clustered mitochondrion. And many rough endocytoplasmic reticulum also appeared in some cells. Some cellular gap connections formed during the induction process. Conclusion Treated with 5-aza, mouse mesenchymal stem ceils may be induced into cardiomyocytes with similar morphology and cardiac specific protein expression.