关键词: 猪, IL-15, 成肌细胞, 细胞凋亡, 细胞周期

Abstract:

【Objective】The aim of this was to investigate the effects of interleukin 15 (IL-15) as a myokine on the proliferation and apoptosis of porcine skeletal muscle myoblast, so as to provide a basis for further studying on the regulation of IL-15 in animal muscle quality and the treatment of skeletal muscle diseases. 【Method】In this study, the IL-15 overexpressed lentiviral vector GV-492-IL-15 was constructed, and the porcine skeletal muscle satellite cells were aseptically isolated and cultured in vitro, then skeletal muscle cells morphology were subjected to myogenic differentiation, and the differentiated myoblast were verified by immunofluorescence staining. After myoblast differentiation, the IL-15 overexpressed recombinant lentiviral vector was transfected. A blank control group (Control), a negative control virus transfection group (IL-15-) and a GV-492-IL-15 lentivirus transfection group (IL-15+) were set for the experiment (n=3). The cells were cultured for 72 h; after growing to a certain number, the cells and the culture supernatant were harvested. Real-time quantitative PCR (qRT-PCR) and Western Blot were used to analyze the expression of target genes and proteins. ELISA kit was used to analyze the content of IL-15 in the culture medium, CCK-8 kit was used to analyze cell viability, and the flow cytometry was used to analyze the results of cell cycle and apoptosis. Moreover, Western Blot was used again to detect changes of the level of caspase-3 protein in the cells, which was closely related to apoptosis.【Result】(1) The identified plasmid was transfected with 293T cells, and as a result, a distinct green fluorescence could be observed in the cells, and a characteristic band near 20 KD could be observed by Western Blot. (2) Fusiform or fusiform porcine skeletal muscle satellite cells were obtained by microscopic observation and differentiated into tubular myoblasts after induction. The differentiated myoblasts were subjected to immunofluorescence staining with α-SMA monoclonal antibody. About 90% of the cells in the visual field were positive, and the cytoplasm stained red, indicating that the cultured cells were skeletal muscle myoblast cells. (3) After transfected with GV-492-IL-15 lentivirus, the relative mRNA and protein expression of IL-15 in myoblasts were significantly increased compared with the control group (P<0.001), however, the protein level of IL-15 in culture medium was not significantly changed (P>0.05). Compared with the control group, the early apoptosis rate of cells transfected with GV-492-IL-15 lentivirus was not significantly different (P>0.05), but the late apoptosis rate of cells was significantly decreased (P<0.05); there was a tendency for caspase-3 protein to decrease compared to the control group, but the overall difference was not significant (P>0.05). The CCK-8 assays showed that overexpression of IL-15 increased the ability of cell proliferation (P<0.05). In addition, the proportion of cells in G1 phase was significantly decreased by transfected with IL-15 overexpression lentivirus, while the proportion of cells in S phase and G2/M phase was significantly increased (P<0.05). 【Conclusion】Under the normal physiological conditions, IL-15 was localized in cells and played a role. The overexpression of IL-15 had no significant effects on the early apoptosis of porcine skeletal muscle myoblasts, but it could inhibit the late apoptosis and promote cell proliferation. This study provided a technical and theoretical basis for the positive regulation of IL-15 on pig skeletal muscle quality and the treatment of related muscle diseases.

Key words: pig, interleukin 15, skeletal muscle myoblast, cell apoptosis, cell cycle