基于转录组学研究双酚A对猪睾丸支持细胞炎症和氨基酸代谢通路的影响

doi:10.11843/j.issn.0366-6964.2023.07.018

摘要/Abstract

摘要： 旨在研究猪睾丸支持细胞（ST）暴露于环境雌激素双酚A （BPA）不同时间后的基因表达谱变化。本研究将猪ST细胞设为C、B两组，每组设置3个重复，C组为空白对照组，B组细胞暴露于50 μmol·L^-1浓度的BPA。BPA暴露6、24 h后，收集C₆、B₆、C₂₄、B₂₄组细胞总RNA样品，构建测序文库，通过双端150 bp测序方法进行高通量转录组测序。对组装数据进行功能注释、差异基因分析以及GO和KEGG富集分析。通过Real-time PCR方法对关键差异基因表达进行验证。BPA暴露24 h后差异基因数量（6 928个）较BPA暴露6 h差异基因数量（3 940个）明显增加。编码分泌型磷蛋白1的SPP1基因在BPA暴露6 h后表达增加，但在BPA暴露24 h后表达降低。管腔结合蛋白编码基因BIP、泛素B编码基因UBB、泛素C编码基因UBC、鸟氨酸脱羧酶1编码基因ODC1表达量在BPA暴露6和24 h后均显著上调。富集分析结果表明，BPA暴露6 h后，TNF信号通路富集差异倍数最高，p53、IL-17以及MAPK信号通路等也均显著富集，暗示ST细胞表现出明显的促炎反应。在BPA暴露24 h后，氨基酸及糖代谢通路富集差异倍数最高，包括精氨酸和脯氨酸、组氨酸以及糖酵解代谢通路。Real-time PCR验证结果显示，BPA暴露6 h后炎性反应通路相关的PTGS2等以及BPA暴露24 h后氨基酸代谢通路相关ARG1等差异基因表达显著上调。综上可知，BPA对猪睾丸支持细胞的损伤机制呈现明显的时间效应，ST细胞在BPA暴露初期表现出以TNF等信号通路激活为特征的炎性反应，在BPA暴露后期氨基酸及糖代谢显著激活。

关键词: 双酚A, 猪睾丸支持细胞, 转录组测序, 富集分析

Abstract: This experiment was conducted to compare the gene expression profile changes of porcine testicular sertoli cells (ST) exposed to environmental estrogen bisphenol A (BPA) for different times. The porcine ST cells were divided into group C and group B with 3 replicates per group. Group C was a blank control group, and group B cells were exposed to BPA at a concentration of 50 μmol·L^-1. At 6 and 24 h after BPA exposure, total RNA samples from C₆, B₆, C₂₄ and B₂₄ group cells were collected, and a sequencing library was constructed. High-throughput transcriptome sequencing was performed using a paired-end 150 bp sequencing method. The assembly data were used for functional annotation, differentially expressed gene (DEG) analysis, and GO and KEGG enrichment analysis. The expression of some key DEGs were validated through real-time PCR method. The number of DEGs (6 928) significantly increased at 24 h after BPA exposure, compared with 3 940 DEGs at 6 h. The expression of SPP1 gene encoding secretory phosphoprotein 1 increased at 6 h after BPA exposure, but decreased at 24 h after BPA exposure. The expression of lumen binding protein-encoding gene BIP, ubiquitin B-encoding gene UBB, ubiquitin C-encoding gene UBC, and ornithine decarboxylase 1-encoding gene ODC1 were significantly upregulated both at 6 and 24 h after BPA exposure. Enrichment analysis showed that the TNF signal pathway was the most significantly enriched at 6 h, and p53, IL-17 and MAPK signal pathways were also significantly enriched, indicating that ST cells exhibited proinflammatory response. At 24 h after BPA exposure, DEGs in pig ST cells were enriched in amino acids and sugar metabolism pathways, including arginine and proline, histidine and glycolysis metabolic pathways. Real-time PCR results showed that PTGS2 and other DEGs related to the inflammatory response pathway at 6 h after BPA exposure and ARG1 and other DEGs related to the amino acid metabolic pathways at 24 h after BPA exposure were significantly up-regulated. In conclusion, the damage mechanism of BPA on porcine sertoli cells shows obvious time effect. Porcine ST cells exhibit inflammatory responses characterized by activation of TNF and other signaling pathways at the early stage of BPA exposure, and the amino acid and glucose metabolism pathways are significantly activated in ST cells at the late stage of BPA exposure.

Key words: bisphenol A, porcine testis sertoli cells, transcriptome sequencing, enrichment analysis

中图分类号:

S828.3

胡婷, 张永红, 侯晓林, 姚华, 崔德凤, 潘早早, 张凌宇, 张家希, 吴琼. 基于转录组学研究双酚A对猪睾丸支持细胞炎症和氨基酸代谢通路的影响[J]. 畜牧兽医学报, 2023, 54(7): 2858-2871.

HU Ting, ZHANG Yonghong, HOU Xiaolin, YAO Hua, CUI Defeng, PAN Zaozao, ZHANG Lingyu, ZHANG Jiaxi, WU Qiong. The Effects of Bisphenol A on Inflammation and Amino Acid Metabolism Pathways in Porcine Testis Sertoli Cells Based on Transcriptome Analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2858-2871.

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