缺氧对鸡成肌细胞肌纤维类型转化作用的机制探究

The Mechanism of Effect of Hypoxia on Myofiber Type Transformation in Chicken Myoblasts

旨在利用氯化钴(CoCl_(2))模拟肌细胞缺氧,并结合转录组测序分析缺氧对肌纤维类型及相关基因的影响。本试验以鸡原代成肌细胞为研究对象,使用不同浓度氯化钴(0、50、100、200和400μmol·L^(-1))处理细胞,每组设3个重复孔。通过检测缺氧标志基因表达和观察细胞形态筛选氯化钴浓度,确定最适宜浓度用于建模。随后,将收集的细胞通过Illumina测序检测缺氧组和对照组鸡成肌细胞的转录表达谱,并分析差异基因和富集到的信号通路。结果表明,200μmol·L^(-1)氯化钴最适合构建鸡胚成肌细胞缺氧模型,通过转录组测序发现,在24和48 h时,缺氧组和对照组分别筛选出1 474个和2 028个差异表达基因,其中有959个差异基因在两个时间点共有。GO和KEGG分析发现,在缺氧环境下,鸡成肌细胞的信号受体活性调节、质膜组成成分、神经活性配体受体作用、PI3K-AKT、肌肉收缩等信号通路关键因子发生明显表达变化。测序和qRT-PCR结果一致显示,缺氧处理后慢肌纤维特异性基因如MYH7B、CSRP2显著上调,而快肌纤维特异性基因如MYH1F、MYH1D、TNNI2、SOX6显著下调,表明缺氧能够诱导鸡成肌细胞快肌纤维向慢肌纤维转换。本研究初步揭示了缺氧对鸡成肌细胞肌纤维类型转换的机制,并提供相关基因表达数据库,为未来家禽肉质和哺乳动物缺氧研究提供了重要参考。

The cobalt chloride(CoCl_(2))was utilized in this study to simulate hypoxia in muscle cells and employed transcriptomic sequencing to analyze the effects of hypoxia on muscle fiber type and genes expression that related to muscle fiber types.Primary chicken myoblasts were treated with different concentrations of CoCl_(2)(0,50,100,200 and 400μmol·L^(-1)),with 3 repeat wells in each group.The CoCl_(2)concentration was screened by detecting the expression of hypoxia marker genes and observing cell morphology to determine the most suitable concentration for modeling.Subsequently,the collected cell was used to detect the gene expression profile of chicken myoblast in hypoxia treatment and control treatment by Illumina technology and assess differential gene expression and enriched signaling pathways between treatments.The results indicated that 200μmol·L^(-1) CoCl_(2)was the most suitable concentration for constructing the hypoxic model in chicken embryonic myogenic cells.Transcriptomic results showed a total of 1474 and 2028 differentially expressed genes in the hypoxia treatment compared to the control group at 24 and 48 h,respectively.Interestingly,959 genes showed differential expression in both time points.GO and KEGG analyses revealed significant alterations in key factors related to signaling pathways such as regulation of signaling receptor activity,integral component of plasma membrane,neuroactive ligand-receptor interaction,PI3K-AKT signaling pathway and muscle contraction in chicken muscle cells under hypoxic conditions.Consistent findings from sequencing and real-time quantitative polymerase chain reaction(qRT-PCR)demonstrated that slow muscle fiber-specific genes such as MYH 7B and CSRP 2 were significantly upregulated after hypoxia treatment,while fast muscle fiber-specific genes including MYH 1F,MYH 1D,TNNI 2,and SOX 6 were significantly downregulated,which indicated a transition of chicken muscle cells from fast to slow muscle fibers was induced by hypoxia.This study provides preliminary insights into the mechanisms underlying the hypoxia-induced transition of muscle fiber types in chicken myogenic cells and offers a valuable gene expression database for future research on poultry meat quality and hypoxia-related studies in mammals.