基于转录组学研究维生素C对猪睾丸细胞增殖活力的影响

Effect of vitamin C on proliferation activity of swine testicular cells based on transcriptome analysis

使用维生素C(VC)处理猪睾丸(ST)细胞,探究其促进ST细胞增殖活力的作用机制,为促进雄性动物生殖系统发育提供依据。通过检测不同浓度VC在不同处理时间下对ST细胞增殖活力的影响以确定VC的最佳作用条件,并检测VC最佳作用条件下对ST细胞增殖活力、细胞内活性氧(ROS)含量、细胞凋亡、细胞周期的影响;根据RNA⁃seq测序数据筛选VC试验组(采用250μmol·L^(-1)VC处理ST细胞72 h)与对照组(采用完全培养基处理ST细胞72 h)ST细胞的差异表达基因,并进行基因本体(GO)功能注释及京都基因与基因组百科全书(KEGG)富集分析。结果表明:(1)用250μmol·L^(-1)VC处理72 h极显著提高了ST细胞增殖活力(P<0.01),且效果最佳;(2)与对照组相比,VC试验组极显著降低了ST细胞内ROS含量、细胞凋亡比例及G0/G1期的细胞比例(P<0.01),同时极显著提高了S期的细胞比例(P<0.01);(3)根据RNA⁃seq测序数据共筛选出1668个差异表达基因,包括1052个上调基因、616个下调基因;(4)GO功能注释和KEGG富集分析显示,差异表达基因多富集在细胞周期、细胞活力、细胞发育、催化活性等生物学过程中,其中,与细胞增殖相关的显著性富集通路包括黏附斑激酶信号通路、FoXO信号通路、磷酸肌醇代谢信号通路等。综上,VC可能通过调控ST细胞周期、凋亡、自噬、代谢的相关基因和信号通路,继而提高ST细胞的增殖活力。

To provide basis for improving the reproductive system development in male animal,the potential mechanism of vitamin C(VC)on promoting the proliferation of swine testicular cell(ST cell)was investigated.The effect of VC concentration and treatment time on ST cell proliferation were firstly studied to obtain the optimal conditions of VC treatment,and then the impact of VC on ST cell proliferation,intracellular reactive oxygen species(ROS)content,cell apoptosis,and cell cycle were evaluated under the opti⁃mal conditions.According to the RNA⁃seq data,the differentially expressed genes(DEGs)between the VC treatment group(50μmol·L^(-1)VC for 72 h)and the control group were screened for subsequent gene ontology(GO)functional annotation and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis.The results showed as follows:(1)treatment with 250μmol·L^(-1)of VC for 72 h significantly increased ST cell proliferation(P<0.01)with the optimal promotive effect.(2)In comparison to the control group,VC treatment significantly reduced ROS content,cell apoptosis,and cell proportion in the G0/G1 phase(P<0.01)while increased cell proportion in the S phase(P<0.01).(3)A total of 1668 DEGs were identified based on RNA⁃seq sequencing data,including 1052 up⁃regulated genes and 616 down⁃regulated genes.(4)GO functional annotation and KEGG enrichment analy⁃sis showed that the DEGs were significantly enriched in biological processes related to cell cycle,cell motility,cell development,catalytic activity,etc.And the significant enrichment pathways related to cell proliferation included focal adhesion kinase signaling pathway,FoXO signaling pathway,and phosphatidylinositol signaling pathway.In conclusion,VC may improve ST cell proliferation by regulating genes and pathways related to cell cycle,apoptosis,autophagy and metabolism.