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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 synthesize three-dimensional polar skeletal muscle tissue with Sylgard 184 groove and C2C12 cells after modification and casting. Method Sylgard 184 bicomponent was evenly mixed in the ratio of 10: 1 and inverted. The plate was allowed to stand at room temperature for curing. The mixture of Hank’s solution, Matrigel and collagen was evenly spread on the bottom of the groove The biological safety cabinet was allowed to dry naturally when the cell matrix was dried. The cells were inoculated with C2C12 cells for more than 1h when the cells were sterilized by UV irradiation. Differentiation medium was induced to differentiate with about 80% confluency. The differentiation status of the myotubes was observed under an inverted microscope. The expressions of myogenin and desmin mRNA in myotubes were detected by PCR, the expression of myogenicin and desmin were detected by immunofluorescence, and the morphology of myotubes and the connections between myotubes were observed by scanning electron microscopy. Results After C2C12 cells were cultured and differentiated for 7 days in the Sylgard 184 elastic mold, the myotube was polarized under the inverted microscope and the confluence of the myotubes was close. After 21 days, the confines of the myotubes were observed by scanning electron microscopy And overlap each other to form a membrane-like structure, the thickness of up to 0.15mm, with three-dimensional; RT-PCR, immunofluorescence test confirmed that myogenicin and desmin in the polarity of myotubes with a positive expression. Conclusion The Sylgard 184 groove modified and cast has a certain directional guiding effect, which can promote the differentiation of C2C12 cells into multi-core myotubes, and the myotubes are arranged in a polar overlap to form a three-dimensional polar skeletal muscle structure.