IL-15过表达对猪骨骼肌细胞成肌分化的影响

Effects of IL-15 Overexpression on Myoblast Differentiation of Porcine Skeletal Muscle Cells

【目的】研究肌肉因子IL-15(interleukin 15)对猪骨骼肌成肌细胞增殖与凋亡的影响,为进一步研究IL-15在动物肌肉品质调控和骨骼肌疾病治疗提供依据。【方法】构建IL-15过表达慢病毒载体GV-492-IL-15,体外无菌分离培养猪骨骼肌卫星细胞,诱导分化,并通过免疫荧光染色进行成肌细胞验证。将成肌细胞转染IL-15过表达重组慢病毒载体,试验分别设置空白对照组(Control)、转染阴性对照病毒组(IL-15-)和转染GV-492-IL-15(IL-15+)慢病毒试验组(n=3)。培养72 h后,收集细胞和培养上清液。分别采用实时定量PCR(qRT-PCR)和Western Blot技术分析目的基因和蛋白的表达情况,采用ELISA试剂盒分析培养液中IL-15含量,采用CCK-8试剂盒分析成肌细胞活力,采用流式细胞术分析细胞周期和细胞凋亡,采用Western Blot技术检测与细胞凋亡密切相关的caspase-3蛋白表达水平的变化。【结果】(1)经鉴定后的质粒转染293T细胞,细胞内可观察到明显的绿色荧光,经Western Blot检测,可以观察到20 kD附近处有特征条带;(2)分离培养的猪骨骼肌卫星细胞呈梭形或纺锤形,诱导后可分化为呈管状的成肌细胞。将分化后的成肌细胞,进行α-SMA单克隆抗体免疫荧光染色,视野中90%的细胞呈阳性反应,胞浆染成红色,表明细胞为骨骼肌成肌细胞。(3)转染GV-492-IL-15慢病毒后,与对照细胞组相比,成肌细胞内IL-15 mRNA和蛋白相对表达量均极显著升高(P<0.001),但培养液中IL-15蛋白水平变化不大(P>0.05)。CCK-8结果显示,过表达IL-15可增强细胞的增殖能力(P<0.05)。与对照组相比,转染GV-492-IL-15慢病毒的细胞早期凋亡率差异不显著(P>0.05),但细胞晚期凋亡率显著下降(P<0.05)。与对照组相比,转染慢病毒组细胞中caspase-3蛋白有下降的趋势,但差异不显著(P>0.05)。此外,转染IL-15过表达慢病毒可使G1期细胞比例显著下降,S期和G2/M期细胞比例显著升高(P<0.05)。【结论】在正常生理条件下,IL-15是定位在细胞内并发挥作用的,IL-15过表达对猪骨骼肌成肌细胞早期凋亡没有显著影响,但可以抑制其晚期凋亡,并促进细胞增殖。这一研究将为IL-15正向调控猪骨骼肌肌肉品质和治疗相关肌肉疾病提供技术和理论依据。

【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-8assays 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.