中国农业科学 ›› 2023, Vol. 56 ›› Issue (2): 391-404.doi: 10.3864/j.issn.0578-1752.2023.02.015

• 畜牧·兽医 • 上一篇    

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

沈龙仙1(),王丽婷1(),何珂1,杜雪1,颜菲菲1,陈维虎2,吕耀平3,汪涵1,周晓龙1(),赵阿勇1()   

  1. 1浙江农林大学动物科技学院/动物医学院,浙江临安 311300
    2宁波市象山县农业农村局,浙江象山 315700
    3丽水学院生态学院,浙江丽水 323000
  • 收稿日期:2021-10-15 接受日期:2022-10-28 出版日期:2023-01-16 发布日期:2023-02-07
  • 通讯作者: 周晓龙,E-mail:zhouxiaolong@zafu.edu.cn。赵阿勇,E-mail:zay503@zafu.edu.cn
  • 作者简介:沈龙仙,E-mail:longxian16@163.com。|王丽婷,E-mail:1365051968@qq.com
  • 基金资助:
    国家自然科学基金(31872397);浙江农林大学学生科研训练项目(S202210341158)

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

SHEN LongXian1(),WANG LiTing1(),HE Ke1,DU Xue1,YAN FeiFei1,CHEN WeiHu2,LÜ YaoPing3,WANG Han1,ZHOU XiaoLong1(),ZHAO AYong1()   

  1. 1College of Animal Science and Technology/College of Veterinary Medicine, Zhejiang A&F University, Lin’an 311300, Zhejiang
    2Xiangshan County Agricultural and Rural Bureau in Ningbo City, Xiangshan 315700, Zhejiang
    3College of Ecology, Lishui University, Lishui 323000, Zhejiang
  • Received:2021-10-15 Accepted:2022-10-28 Online:2023-01-16 Published:2023-02-07

摘要:

【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1 μg·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(MTNR1AMTNR1B)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 blot实验技术研究过表达MTNR1AMTNR1B是否影响MLT对增殖基因的促进作用;为了进一步探究MLT与NMN对鹅骨骼肌卫星细胞增殖相关基因的影响,采用qRT-PCR和Western blot实验技术检测骨骼肌细胞增殖相关基因的表达变化。【结果】骨骼肌卫星细胞的特异性标志蛋白Pax7和Desmin分别在细胞核和细胞质中呈绿色荧光的阳性反应,结果表明试验所用细胞为骨骼肌卫星细胞,CCK-8检测结果表示1 ng·mL-1 MLT与1 μg·mL-1 NMN促进鹅骨骼肌卫星细胞活性,qRT-PCR和Western blot结果显示,与对照组相比,过表达MLT受体基因MTNR1A、MTNR1B后,增殖标志基因Pax7的mRNA和蛋白表达显著上调(P<0.05),抑制增殖的标志基因MSTN的mRNA和蛋白表达显著下调(P<0.05);另外,1 ng·mL-1 MLT和1 μg·mL-1 NMN共同处理鹅骨骼肌卫星细胞后,增殖标志基因Pax7的mRNA和蛋白表达显著上调(P<0.05),抑制增殖的标志基因MSTN的mRNA和蛋白表达显著下调(P<0.05),其变化比NMN和MLT单独处理更为显著。【结论】MLT通过其受体促进骨骼肌卫星细胞增殖,NMN和MLT共同处理可以加强MLT对鹅骨骼肌卫星细胞增殖的促进作用,也为MLT和NMN在家禽实际生产中的应用提供了新思路。

关键词: 浙东白鹅, 褪黑素, 烟酰胺单核苷酸, 过表达载体, 骨骼肌

Abstract:

【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.

Key words: East Zhejiang White Goose, melatonin, nicotinamide mononucleotides, overexpression vector, skeletal muscle