褪黑素和烟酰胺单核苷酸对鹅骨骼肌卫星细胞增殖的影响

【背景】目前关于褪黑素（melatonin,MLT）的研究多集中在家禽的生殖功能上，而与家禽肌肉发育相关的作用机制却鲜有研究。同时MLT与烟酰胺单核苷酸(nicotinamide mononucleotide, NMN)功能相似且都与昼夜节律相关，研究发现MLT与NMN的单一处理对细胞衰老的影响有限，而共同处理的效果更为显著，虽然二者对线粒体功能和骨骼肌衰老的影响也有相关报道，但在骨骼肌生长发育的作用机制方面尚无可以参考的报道。【目的】通过探究MLT与NMN参与浙东白鹅骨骼肌卫星细胞增殖的分子机制，为其在家禽生产实践中的应用提供思路。【方法】通过解剖鹅胚分离培养鹅骨骼肌卫星细胞，并对骨骼肌卫星细胞的特异性蛋白Pax7和Desmin进行免疫荧光染色以此鉴定细胞，在体外成熟培养基础上用1 ng·mL-1 MLT与1μg·mL-1 NMN分别单独或组合处理细胞24 h后，利用CCK-8检测细胞活力；为探究MLT如何调控鹅骨骼肌卫星细胞增殖的机制，通过构建MLT受体基因（MTNR1A、MTNR1B）的过表达载体，并与1 ng·mL-1 MLT共同处理细胞，采用qRT-PCR和Western b...

Effects of Melatonin and Nicotinamide Mononucleotides on Proliferation of Skeletal Muscle Satellite Cells in Goose

【Background】 At present, the research on melatonin (MLT) is mostly focused on the reproductive function of poultry, and there is little research on the mechanism of action related to muscle development in poultry. At the same time, MLT and nicotinamide mononucleotide (NMN) have similar functions and are related to circadian rhythm. The previous study found that the single treatment of MLT and NMN had limited effect on cell senescence, while the effect of co-treatment was more significant, there are also related reports on the effects of skeletal muscle on mitochondrial function and skeletal muscle aging, but there is no report on the mechanism of skeletal muscle growth and development. 【Objective】 This study aimed to explore the molecular mechanism of MLT and NMN participating in the proliferation of skeletal muscle satellite cells of East Zhejiang White Goose. 【Method】 The goose skeletal muscle satellite cells were isolated and cultured by dissecting goose embryos, and the specific proteins Pax7 and Desmin of skeletal muscle satellite cells were subjected to immunofluorescence staining to identify cells. The cells were treated with 1 ng·mL^-1 MLT and 1 μg·mL^-1 NMN alone or in combination for 24 h in vitro mature culture, and then CCK-8 was used to detect the cell viability. In order to explore the clear mechanism of how MLT regulated the proliferation of goose skeletal muscle satellite cells, MLT receptor genes (MTNR1A, MTNR1B) were constructed, and then the cells were co-treated with 1 ng·mL^-1 MLT, after that qRT-PCR and Western blot experiments were used to study whether overexpression of MTNR1A and MTNR1B affected the promotion of MLT on proliferation genes. In order to further explore the effects of MLT and NMN on the goose skeletal muscle satellite cell proliferation-related genes were detected by qRT-PCR and Western blot experiments to detect the expression changes of skeletal muscle cell proliferation-related genes. 【Result】 Pax7 and Desmin who were the specific marker proteins of skeletal muscle satellite cells were positive for green fluorescence in the nucleus and cytoplasm. The results showed that the cells used in the experiment were skeletal muscle satellite cells. The results of CCK-8 assay indicated that 1 ng·mL^-1 MLT and 1 μg·mL^-1 NMN could promote the activity of goose skeletal muscle satellite cells. The results of qRT-PCR and Western blot showed that compared with the control group, after overexpressed MLT receptor gene MTNR1A and MTNR1B, the mRNA and protein expressions of the proliferation marker gene Pax7 were significantly up-regulated (P<0.05), and the mRNA and protein expressions of the proliferation-inhibiting marker gene MSTN were significantly down-regulated (P<0.05). In addition, after co-treatment of goose skeletal muscle satellite cells with 1 ng·mL^-1 MLT and 1 μg·mL^-1 NMN, the mRNA and protein expressions of the proliferation marker gene Pax7 were significantly up-regulated (P<0.05), and the mRNA and protein expressions of the proliferation-inhibiting marker gene MSTN were significantly down-regulated (P<0.05), the change of which was more significant than that of NMN and MLT alone. 【Conclusion】 MLT promoted the proliferation of skeletal muscle satellite cells through its receptors, and the co-treatment of NMN and MLT could enhance the promoting effect of MLT on the proliferation of goose skeletal muscle satellite cells, and also provided a new idea for the application of MLT and NMN in the actual production of poultry.