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

doi:10.3864/j.issn.0578-1752.2023.02.015

摘要/Abstract

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（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 blot实验技术研究过表达MTNR1A、MTNR1B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

沈龙仙, 王丽婷, 何珂, 杜雪, 颜菲菲, 陈维虎, 吕耀平, 汪涵, 周晓龙, 赵阿勇. 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[J]. 中国农业科学, 2023, 56(2): 391-404.

SHEN LongXian, WANG LiTing, HE Ke, DU Xue, YAN FeiFei, CHEN WeiHu, LÜ YaoPing, WANG Han, ZHOU XiaoLong, ZHAO AYong. Effects of Melatonin and Nicotinamide Mononucleotides on Proliferation of Skeletal Muscle Satellite Cells in Goose[J]. Scientia Agricultura Sinica, 2023, 56(2): 391-404.

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名称 Name	引物序列 Primer sequence (5′—3′)	用途 Purpose	参考序列 Reference sequence
Pax7	F：CCTGGGCGACAAAGGTAA	qRT-PCR	XM_013187867.1
Pax7	R：GCTCAGCGGTGAAAGTGG	qRT-PCR	XM_013187867.1
MyoD	F：GGCTCAGCAAGGTCAACG	qRT-PCR	XM_013177726.1
MyoD	R：TCCAGCACCGGGTAGTAAA	qRT-PCR	XM_013177726.1
Myf5	F：GAGGAGGCTGAAGAAAGTGAA	qRT-PCR	XM_013195729.1
Myf5	R：GCTCTGTCCAGGCAGGTGAT	qRT-PCR	XM_013195729.1
MSTN	F：GGCTCTTGATGACGGTAG	qRT-PCR	XM_013178647.1
MSTN	R：CTTGTTCCAGACGCAGTT	qRT-PCR	XM_013178647.1
MyoG	F：CGCCTGAAGAAGGTGAACGAAGC	qRT-PCR	XM_013196590.1
MyoG	R：GTCCCTCTGCTCCCGCTCCTG	qRT-PCR	XM_013196590.1
Myf6	F：AGCAGGCAAATGGCTCGGACTTC	qRT-PCR	XM_013195738.1
Myf6	R：GCTTGGGCTCGTCGGAGGAAAT	qRT-PCR	XM_013195738.1
Pax3	F：AGCCATCCTACCAGCCCACCTC	qRT-PCR	XM_013181590.1
Pax3	R：CGAAGGGAGGCTGCTTTGGTGT	qRT-PCR	XM_013181590.1
GAPDH	F：AGCTGATCTCCCATGTTCGTG	内参基因	MG674174.1
GAPDH	R：GCTCCCTCCACAATGCCAAAG	内参基因	MG674174.1

抗体 Antibody	货号 Article number	厂家 Manufacturer	稀释倍数 Dilution ratio
Pax7	ab187339	Abcam, 英国 England	1:1000
MSTN	A6913	ABclonal, 中国 China	1:1000
MyoD1	A16218	ABclonal, 中国 China	1:1000
β-actin	AC026	ABclonal, 中国China	1:1000
山羊抗兔HRP, Goat anti rabbit IgG	A21020	Abbkine, 瑞士 Switzerland	1:10000