去乙酰化酶Sirt1对牛骨骼肌卫星细胞增殖和分化的影响

doi:10.11843/j.issn.0366-6964.2025.02.012

摘要/Abstract

摘要：

旨在探讨沉默信息调节因子2相关酶1(silent information regulator 2-related enzyme1, Sirt1)对牛骨骼肌卫星细胞(bovine skeletal muscle satellite cells, BSMSCs)增殖和成肌分化的影响。在本试验中，将小片段干扰RNA(siRNA)和靶向Sirt1的过表达质粒转染入从3~4月龄胎牛臀腿肌剥离的原代卫星细胞中，在增殖和分化阶段的特定时间点收集RNA和蛋白质样品，每个处理设计3个生物学重复，使用实时定量PCR(qRT-PCR)以及蛋白质印记法(Western blotting)进行分析。此外，还进行了EdU染色试验以评估细胞的增殖情况。结果显示：在细胞分化期，40倍显微镜下观察，发现Sirt1敲低导致肌管的数量、长度和直径显著增加。qRT-PCR和Western blotting证实，与对照组相比，分化标志物肌球蛋白重链(myosin heavy chains, MyHC)和肌细胞生成素(myogenin, MyoG)的mRNA水平显著上调(P < 0.01)，MyoG的蛋白表达水平也显著升高(P < 0.05)。相反，Sirt1过表达导致肌管的数量、长度和直径显著减少。Western blotting显示Sirt1过表达组MyoG表达量显著低于对照组(P < 0.01)。在BSMSCs增殖期，qRT-PCR、Western blotting和EdU检测表明，Sirt1敲低和过表达对细胞增殖均无显著影响(P>0.05)。综上所述，Sirt1敲减可有效促进细胞分化，而过表达可抑制该过程，但对增殖均无明显影响。这些发现证实了Sirt1是作为调控BSMSCs成肌分化的重要因素，在协调成肌细胞分化和促进肌管形成中起关键作用。

关键词: Sirt1, 牛骨骼肌卫星细胞, 细胞增殖, 成肌分化

Abstract:

The aim of this study was to investigate the effect of silent information regulator 2-related enzyme1 (Sirt1) on proliferation and myogenic differentiation of BSMSCs. In this experiment, small fragments of interfering RNA (siRNA) and overexpressed plasmid targeting Sirt1 were transfected into primary satellite cells stripped from the thigh muscle of 3-4-month-old fetal cattle. RNA and protein samples were collected at specific time points during the proliferation and differentiation stages, with 3 biological replicates per treatment. Real-time quantitative PCR (qRT-PCR) and Western blotting were used for analysis. In addition, EdU staining experiments were performed to evaluate cell proliferation.The results showed that the number, length, and diameter of muscle ducts were significantly increased by Sirt1 knockdown under a 40-fold microscope during cell differentiation. qRT-PCR and Western blotting confirmed that compared with the control group, mRNA levels of differentiation markers myosin heavy chains (MyHC) and myogenin (MyoG) were significantly up-regulated after interfering Sitr1 (P < 0.01). The protein expression level of MyoG was also significantly increased (P < 0.05) after interfering Sitr1. Conversely, Sirt1 overexpression resulted in a significant reduction in the number, length, and diameter of muscle ducts. Western blotting showed that the expression of MyoG in Sirt1 overexpression group was significantly lower than that in control group (P < 0.01). At the proliferation stage of BSMSCs, qRT-PCR, Western blotting and EdU tests showed that Sirt1 knockdown and overexpression had no significant effects on cell proliferation (P>0.05). In summary, Sirt1 knockdown can effectively promote cell differentiation, while overexpression can inhibit the process, but has no significant effect on proliferation. These findings confirm that Sirt1 is an important factor regulating the myogenic differentiation of BSMSCs, and plays a key role in coordinating myoblast differentiation and promoting myotube formation.

Key words: Sirt1, bovine skeletal muscle satellite cells, cell proliferation, myogenic differentiation

中图分类号:

S823.2

张正雨, 杨培鸿, 郭宏, 李新, 张林林, 郭益文, 胡德宝, 丁向彬. 去乙酰化酶Sirt1对牛骨骼肌卫星细胞增殖和分化的影响[J]. 畜牧兽医学报, 2025, 56(2): 603-610.

ZHANG Zhengyu, YANG Peihong, GUO Hong, LI Xin, ZHANG Linlin, GUO Yiwen, HU Debao, DING Xiangbin. Effects of Sirt1 Deacetylase on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 603-610.

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引物名称 Primers name	基因ID GenBank ID	引物序列(5′→3′) Sequence	产物大小/bp Products size
Sirt1 forward	NM_001192980.3	CATTTTCCATGGCGCTGAGG	104
Sirt1 reverse	NM_001192980.3	AACTTGGACTCTGGCACGTT	104
GAPDH forward	NM_001034034.2	CCGCATCCCTGAGACAAGAT	114
GAPDH reverse	NM_001034034.2	ATTGATGGCGACGATGTCCA	114
Pax7 forward	XM_015460690.3	CGATTAGCCGAGTGCTCAGA	137
Pax7 reverse	XM_015460690.3	GTCCAGACGGTTCCCTTTGT	137
Ki67 forward	NM_001034354.2	CCTAAACCCGCAGGAGGATG	78
Ki67 reverse	NM_001034354.2	TTCTCCTGTTGCTTGGTCGC	78
MyoG forward	NM_001111325.1	GTGGGCGTGTAAGGTGTGTA	91
MyoG reverse	NM_001111325.1	CACCTTCTTGAGTCTGCGCT	91
MyHC forward	NM_174117.1	TCAAGGTTGCATCTCCAAGGC	131
MyHC reverse	NM_174117.1	GCGCTCCTTTTCAGACTTTCG	131

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