C2C12细胞诱导构建三维骨骼肌组织

Construction of highly organized three-dimensional muscle tissue induced by C2C12 cells in vitro

目的利用修饰并铸型后的Sylgard 184凹槽与C2C12细胞复合培养、诱导分化,获取三维极性骨骼肌组织。方法 Sylgard 184双组分以10∶1的比例均匀混合并倒板,室温下静置固化并对其表面压槽铸型,Hank液冲洗凹槽,Matrigel和胶原的混合液均匀铺被凹槽底部,置生物安全柜待细胞基质自然干燥、紫外线照射消毒1h以上时接种C2C12细胞悬液,细胞增殖约80%汇合时改用分化培养基进行分化诱导,倒置显微镜下观察肌管的分化状态,RT-PCR方法检测肌管内myogenin和desmin基因mRNAs的表达,免疫荧光检测生肌转录因子myogenin和desmin蛋白的表达,扫描电镜观察肌管形态和肌管间的连接。结果 C2C12细胞在Sylgard 184弹性体铸型压槽中培养分化7d后,倒置显微镜下可见肌管呈极性分化,且肌管之间融合紧密;21d后,扫描电镜检测可见肌管之间排列紧密且相互重叠,形成膜样结构,厚度可达0.15mm,具有三维性;RT-PCR、免疫荧光检测证实极性分化肌管内具有myogenin和desmin的阳性表达。结论修饰并铸型的Sylgard 184凹槽具有一定的方向引导效应,能促进C2C12细胞分化形成多核肌管,且肌管呈极性重叠排列,形成三维极性骨骼肌组织结构。

Objective To cultivate C2C12 cells on the modified mold-grooves of Sylgard 184 and induce C2C12 cells differentiating into highly organized three-dimensional muscle tissue. Methods Bicomponents Sylgard 184 was mixed uniformly by the ratio of 10：1 and poured into 6-well plates. Solidified by standing at room temperature, the surface of Sylgard 184 mold-grooves was casted and washed with Hank solution. Matrigel matrix and collagen were mixed uniformly, then paved at the bottom of grooves and airdried in the biological safety cabinet , making sure that the cell matrix material of coverage was uniform. After UV disinfection, C2C12 cells suspension was inoculated. When cells reached to 80% confluence, then C2C12 cells were differentiation cultivated. The morphology of myotubes was observed under the inverted microscopy. RT-PCR was used to test expression of myogenin and desmin mRNA in skeletal muscle tissue. Expression of myogenin and desmin muscle-specific protein was tested by immunofluorescence. Morphologies and inter-connections of myotubes were observed under scanning electronic microscope. Results As C2C12 cells were differentiation cultivated in the mold-grooves of Sylgard 184 for 7 days, differentiation polarity and myotube fusion were observed under the inverted microscope. After 21 days, it showed that myotubes stood closely and connected with each other detected by the scanning electron microscopy. The thickness of the membrane-like structure with the character of three-dimensional was 0. 15mm. The positive expressions of myogenin and desmin mRNAs were detected in skeletal muscle tissue by RT-PCR. Immunofluorescence and DAPI nuclear staining showed that myogenin and desmin gene protein were expressed positively. Conclusion The modified mold-grooves of Sylgard 184 is able to guide the differentiation of C2C12 cells and promotes them to form parallel muhinucleated myotubes . Myotubes could overlap with each other and form threedimensional skeletal muscle tissue.