Abstract:

Objective To establish a model for in vitro culture and differentiation of skeletal muscular cells and identification of muscular fibers. Methods C2C12 cells of skeletal muscle were obtained, 5×10⁴/mL C2C12 cells were cultured in DMEM with 20 mL/L horse serum in a incubator for in vitro differentiation. The expression of myogenin was detected by immunofluorescence technique. The differentiated C2C12 cells were treated with 25 ng/mL visfatin for 48 h (visfatin treatment group) or not treated with visfatin (control group), the expression of mRNA and protein of myosin heavy chain (MHC) subtypes (MHC-Ⅰ, MHC-ⅡA and MHC-ⅡB) was detected by RT-PCR and Western blotting, and the ratios of expression of MHC-Ⅰ to MHC-Ⅱ were calculated. Results The undifferentiated spindle-like cells had only one nucleus with several nucleoli, while the differentiated cells became elongated with numerous nuclei clustering like a string. Immunofluorescence detection demonstrated that myogenin was expressed in the nucleus which confirmed the differentiation of C2C12 cells. RT-PCR and Western blotting indicated the capability of C2C12 cells to express mRNA and protein of three MHC subtypes after in vitro induced differentiation, and the expression of mRNA and protein of three MHC subtypes in visfatin treatment group was significantly higher than that in control group (P<0.05). The ratios of expression of MHC-Ⅰ to that of MHC-Ⅱ in control group and visfatin treatment group were 0.63 and 0.60 respectively. Conclusion C2C12 cell model can be used to induce the differentiation of skeletal muscle and identify the types of muscular fibers, and the cell model responses to visfatin at its lower concentration, which provides basis for the further research on the mechanism of upper airway collapse.

Key words: C2C12 cells, differentiation, myogenin, myosin heavy chain, identification of skeletal muscular fibers