小鼠骨髓间充质干细胞体外诱导向心肌细胞分化的研究

Study on the Induced Differentiation of Mouse Mesenchymal Stem Cells into Cadiocytes in Vitro

目的探讨体外诱导小鼠骨髓间充质干细胞(MSCs)向心肌细胞分化的方法。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代细胞培养48h,分为4组:A组加入心肌组织块条件培养液、B组加10μmol/L 5-氮胞苷(5-aza)、C组为二者的混合液、D组不加任何诱导剂以作对照组。应用免疫细胞化学方法检测各组心肌特异性蛋白α-actin的表达。结果免疫细胞化学染色结果显示,A组细胞培养3周时可检测到α-actin阳性细胞,阳性细胞较多;B组培养1周可见弱阳性细胞,随时间延长,阳性着色增强且阳性细胞数增多,3周时呈强阳性表达;C组2周即可检测到细胞呈强阳性着色。对照组MSCs胞浆内未检测到α-actin的表达。4个组的阳性细胞率进行两两比较均有统计学意义(P<0.05)。结论心肌组织块条件培养液和5-aza联合应用可促进MSCs体外分化为心肌样细胞。

Objective To investigate the method of induecd differentiation of mouse mesenchymal stem cells into eadioeytes in vitro.Methods The mononuclear cells were isolated from tihias and fibulas of 2 weeks old mice and the mesenehymal stem cells of bone marrow were oblained with the combination of gradient centrifugation and cellular attaching culture method. Cells of the 2nd passage were divided into 4 groups at 48h,cells of group A were treated with simple upper liquid from cardiac tissue cuhure. 10μmol/L 5-aza was added to group B. In group C,cells were given assuciated treatment with the two factors. Group D was given no inductor for eonh＇ol. Then the expression of α-actin from the 4 groups was detected hy immunoeytochemistry method. Results Cells treated with cardiac tissue upper liquid （ group A ） for 3 weeks showed a positive expression of α-actin. With treatment of 101μmol/L 5-aza （ group B）at 3 weeks, cells were strongly positive stained. In group C,cells took on intensively positive staining at 2 weeks afler co-treated with 101μmol/L 5-aza and cardiac tissue euhure upper liquid and there was no positive cell all the time in group D. Comparison between every two groups among A,B,C and l） showed statistical difference（P 〈0.05 ）. Conclusion Co-treatment of cardiac tissue euhure upper liquid and 10μmol/L 5-aza may aeeelerate the differentiation from mesenchymal stem cells into eadiocytes in vitro.