牵拉应力在5-氮杂胞苷诱导骨髓间充质干细胞成肌过程中的动态效应

Dynamic effects of stretch stress on bone marrow mesenchymal stem cell differentiation into myoblasts induced by 5-azacytidine

背景：国内外研究发现在一定力学环境下，体外培养的间充质干细胞其增殖分化等一系列生物学特性会发生相应变化。目的：体外观察5-氮杂胞苷与牵拉应力对大鼠骨髓间充质干细胞向肌细胞分化的调控作用。设计、时间及地点：2006-01／2007-06在上海交通大学生物力学实验室完成的细胞观察实验。材料：清洁级3—4周龄雄性SD大鼠30只用于分离培养骨髓间充质干细胞，1-3d龄SD新生鼠4只用于建立骨骼肌阳性细胞株。化学诱导剂5-氮杂胞苷为Sigma公司产品，牵拉设备FX-4000 Flexercell由美国Flexceu Int公司生产。方法：将传至第4代的骨髓间充质干细胞接种于Flex6孔板。①设立5组行成肌家族因子表达Westem—Blot检测：第1组细胞单纯经10μmol/L5-氮杂胞苷诱导1周；第2—4组细胞以10μmol/L5-氮杂胞苷诱导1周后，在1Hz频率、15％幅度应力条件下分别牵拉12，24，36h；第5组以新生鼠骨骼肌细胞作为阳性对照。②设立5组行诱导剂及牵拉应力作用的RT—PCR检测：第1组细胞给予1Hz频率、15％幅度应力牵拉24h；第2、3组分别经10μmol/L5-氮杂胞苷诱导1，2周；第4组细胞经10μmol/L5-氮杂胞苷诱导2周后，给与同等应力牵拉24h；第5组以新生鼠骨骼肌细胞作为阳性对照。主要观察指标：不同诱导条件及应力牵拉状态下骨髓间充质干细胞成肌家族因子的表达。结果：①第1组表达成肌因子MyoD与Myf5，成熟肌细胞标志MHC表达不显著；第2—4组MyoD与Myf5的表达明显增强（P〈0．05），且随牵拉时间延长因子的表达逐步增强，MHC表达不显著。②第1组不表达成肌家族因子；第2组表达MvoD、Myf5，第3组表达MyoD、Myf5、desmin，第4组几乎表达所有检测的肌细胞特异性标记。结论：经5氮杂胞苷诱导可启动骨髓间充质干细胞向肌细胞分化，单独的牵拉应力不能诱导成肌。牵拉应力可调控5氮杂胞苷对骨髓间充质干细胞的诱导分化进程，并且与牵拉作用时间成正性相关。

BACKGROUND： In vitro cultured biological characteristics of mesenchymal stem cells （MSCs） changed under a mechanics environment such as proliferation or differentiation. OBJECTIVE： To observe the regulatory effects of stretch stress on rat bone marrow mesenchymal stem cell （BMSC） differentiation into myoblasts induced by 5-azacytidine, DESIGN, TIME AND SETTING： The cell experiment was conducted at the Biomechanics Laboratory of Shanghai Jiao Tong University from January 2006 to June 2007. MATERIALS： Totally 30 male SD rats aged 3-4 weeks of clean grade were selected for BMSC culture. Four neonatal SD rats aged 1-3 days were selected for establishing skeletal muscle positive cell strain. 5-azacytidine was purchased from Sigma, USA and FX-4000 Flexercell was bought from Flexcell Int, USA, METHODS： Fourth passage of BMSCs was inoculated on the Flex 6-well plate, Western-blot： BMSCs in the group 1 were induced by 10 μ mol/L 5-azacytidine for 1 week. BMSCs in the groups 2-4 were induced by 10 μ mol/L 5-azacytidine for 1 week, and then dragged at a frequency of 1 Hz and an extent of 15% for 12, 24 and 36 hours respectively. Skeletal muscles from neonatal rats in the group 5 were selected as a positive control, Reverse transcriptase-polymerase chain reaction （RT-PCR）： BMSCs in the group 1 were dragged at a frequency of 1 Hz and an extent of 15% for 24 hours, BMSCs in the groups 2 and 3 were induced by 101a mol/L 5-azacytidine for 1 and 2 weeks, respectively, BMSCs in the group 4 were induced by 10 μ mol/L 5-azacytldine for 2 weeks, and then dragged at a frequency of 1 Hz and an extent of 15% for 24 hours. Skeletal muscles from neonatal rats in the group 5 were selected as a positive control. MAIN OUTCOME MEASURES： Myogenic family factors of BMSCs under different induction and stress conditions, RESULTS： MyoD and Myf5 were positive, but MHC was negative in the group 1. MyoD and Myf5 levels were increased in the groups 2-4 （P 〈 0.05）, and increased by prolongation of dragged time, MHC expression was not significant, Myogenic family factors were not detected in the group 1. MyoD and Myf5 were positive in the group 2, MyoD, Myf5 and desmin were positive in the group 3. All myoblast specific markers were found in the group 4. CONCLUSION： 5-azacytidine can induce the differentiation of BMSCs into myoblasts. Stretch stress alone cannot induce myoblasts. Stretch stress also can adjust the induction of 5-azacytidine on BMSCs, and is positively correlated with stretch time.