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旨在研究TG结合因子1(TG-interacting factor 1, Tgif1)对猪骨骼肌卫星细胞(muscle satellite cells, MuSCs)增殖与分化能力的影响。本研究利用CRISPR/Cas9基因编辑技术在MuSCs中沉默Tgif1基因，并利用Western blot和荧光定量PCR(qPCR)验证基因编辑效率；采用免疫荧光染色、Western blot、qPCR检测细胞分化能力，5′-乙炔基-2′-脱氧尿苷(EdU)、细胞计数试剂盒-8(CCK-8)评估沉默Tgif1基因对MuSCs增殖能力的影响；进一步结合转录组测序技术(RNA-seq)对沉默Tgif1基因的MuSCs的基因表达模式变化进行分析，鉴别出了差异表达基因(differential expressed genes, DEGs),并进行了基因本体(GO)和京都基因组百科全书(KEGG)通路富集分析。结果：缺失Tgif1基因会显著抑制肌管的生成(P<0.01),但对MuSCs增殖能力没有显著影响(P>0.05)。RNA-seq分析显示，Tgif1基因缺失导致434个基因发生了显著变化，包括72个显著上调表达基因和362个显著下调表达基因，其中包括作用于成肌细胞分化早期并正向调控肌生成的肌生成分化因子1(myoblast determination protein, MyoD1),胆碱能受体烟碱α1亚单位(cholinergic receptor nicotinic alpha 1 subunit, Chrna1),早期生长反应基因1(early growth response 1, Egr1)和双特异性磷酸酶1基因(dual specificity phosphatases 1, Dusp1),这些DEGs主要富集在细胞迁移功能条目以及cAMP、PI3K-Akt和钙信号等信号通路中。综上，本研究证明了Tgif1是一个正向调控猪骨骼肌生成的关键因子，研究结果为猪骨骼肌生长发育的遗传解析提供了新的理论依据，并为产肉性状的精准遗传调控提供了新的候选靶点。
Abstract：The aim of this study was to investigate the effects of TG-interacting factor 1(Tgif1) on the proliferation and differentiation capabilities of porcine skeletal muscle satellite cells(MuSCs). CRISPR/Cas9 gene editing technology was employed to silence the Tgif1 gene in porcine muscle satellite cells, with gene editing efficiency validated via Western blot and quantitative real-time PCR(qPCR). Then, immunofluorescence staining, Western blot, and qPCR were employed to assess cellular differentiation capacity. Next, 5′-Ethynyl-2′-deoxyuridine(EdU) and the Cell Counting Kit-8(CCK-8) were used to evaluate the impact of Tgif1 silencing on MuSCs proliferation. Finally, further analysis of gene expression patterns in Tgif1-silenced MuSCs was performed using RNA-sequencing(RNA-seq), identifying differentially expressed genes(DEGs) and conducting Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The results showed that Tgif1 gene deletion significantly inhibited myotube formation(P<0.01) but had no significant effect on cell proliferation capacity(P>0.05). RNA-seq analysis revealed that Tgif1 deletion induced significant changes in 434 genes, including 72 significantly upregulated and 362 significantly downregulated genes. Among these were myoblast determination protein 1,the cholinergic receptor nicotinic alpha 1 subunit,early growth response 1,and dual specificity phosphatase 1,which acted early in myocyte differentiation and positively regulates myogenesis. These DEGs were predominantly enriched in cell migration-related functional categories and signaling pathways such as c AMP,PI3K-Akt,and calcium signaling. In summary,this study indicated that Tgif1 was a key factor positively regulating porcine skeletal muscle development. These findings provided new theoretical support for the genetic analysis of porcine skeletal muscle growth and development,and offered novel candidate targets for the precise genetic regulation of meat production traits.
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基本信息:
中图分类号:S828
引用信息:
[1]赵赶,甘秀娟,倪梦茹,等.转录因子Tgif1对猪骨骼肌卫星细胞增殖与分化能力的影响[J].畜牧与兽医,2025,57(12):17-26.
基金信息:
国家自然科学基金项目(32172710); 江苏省种业振兴工程项目(JBGS[2021]024、JBGS[2021]026); 江苏省农业产业技术体系项目(JATS[2023]410、JATS[2023]186); 常州市生猪种业发展创新中心项目(CAIC[2024]001)