文献类型：期刊文献

中文题名：基于转录组分析CSFV感染猪睾丸支持细胞对屏障功能相关基因的影响

英文题名：Transcriptomic Analysis of the Effects of CSFV Infection on Barrier Function-Related Genes in ST Cells

作者：马尧丹[1];俞晖[1];陈海恩[1];杨阳开心[1];葛书君[1];吴江[1];康恺[1]

机构：[1]广东海洋大学滨海农业学院,广东湛江524088

年份：2025

卷号：61

期号：5

起止页码：22

中文期刊名：中国兽医杂志

外文期刊名：Chinese Journal of Veterinary Medicine

收录：北大核心2023、、北大核心

基金：广东省自然科学基金(2020A1515110362);广东省科技厅海外名师项目(K23438);湛江市科学技术局海洋生物方向基金(2021E05028)。

语种：中文

中文关键词：猪瘟病毒(CSFV);猪睾丸支持细胞;mRNA差异表达;基因本体论(GO)功能注释;京都基因与基因组百科全书数据库(KEGG)富集分析

外文关键词：classical swine fever virus(CSFV);sertoli cells;differential mRNA expression;Gene ontology(GO)function annotation;Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis

中文摘要：为探究猪瘟病毒(CSFV)感染对猪睾丸支持细胞(ST)mRNA表达谱的影响,本试验以感染CSFV 24和48 h的ST细胞和未感染ST细胞为模型,通过蛋白免疫印迹试验(WB)和间接免疫荧光(IIF)染色检测CSFV E2蛋白的表达;构建4个cDNA文库进行转录组测序,对差异表达基因(DEGs)进行基因本体论(GO)功能注释和京都基因与基因组百科全书数据库(KEGG)富集分析并筛选血睾屏障相关DEGs,通过实时荧光定量聚合酶链式反应(qPCR)、WB和IIF对DEGs进行验证。结果显示,CSFV感染24和48 h后,ST细胞均表达了E2蛋白;与Control 24 h相比,CSFV 24 h的ST细胞共鉴定出86个显著差异DEGs(P <0.05);与Control 48 h相比,CSFV 48 h的ST细胞共鉴定出1 481个显著差异DEGs(P <0.05)。DEGs极显著富集在27个GO功能条目上,主要包括生物学过程、细胞组分和分子功能(P <0.01);极显著富集的KEGG信号通路主要包括肿瘤坏死因子(TNF)信号通路、晚期糖基化终产物受体(AGE-RAGE)信号通路、白细胞介素-17(IL-17)信号通路和丝裂原活化蛋白激酶(MAPK)信号通路等(P <0.01)。DEGs的qPCR验证结果显示,与Control 24 h相比,CSFV 24 h的CLDN11、H2AFX和CDH2表达量极其或极显著上调(P<0.001或P <0.01),TJP1、OCLN和GJA1表达量显著下调(P <0.05);与Control48 h相比,CSFV 48 h的FASLG、TGFB1、IL-6和H2AFX表达量极其或极显著上调(P <0.001或P <0.01),TJP1、OCLN、GJA1、CLDN11和CDH2表达量极显著下调(P <0.01)。差异表达蛋白WB验证结果显示,与Control 24 h相比,CSFV 24 h的TJP1、CLDN11和GJA1蛋白表达量极其或极显著下调(P <0.001或P <0.01),CLDN11蛋白表达量极其显著上调(P <0.001);与Control 48 h相比,CSFV 48 h的TJP1、CLDN11、OCLN和GJA1蛋白表达量极其或极显著下调(P <0.001或P <0.01)。IIF结果显示,与Control 48 h相比,CSFV 48 h的TJP1蛋白相对荧光面积极显著下调(P <0.01),CLDN11、OCLN和GJA1蛋白相对荧光面积显著下调(P <0.05)。以上验证结果与转录组测序结果基本一致。结果表明,CSFV感染24和48 h均会影响ST细胞屏障功能相关基因的表达,本试验为进一步探究CSFV对ST细胞屏障的影响机制提供了数据支持。

外文摘要：To investigate the impact of classical swine fever virus(CSFV)infection on the mRNA expression profile of swine testicular Sertoli cells(ST),ST cells infected with CSFV for 24 h and 48 h,along with uninfected control cells,were used as models.Western blotting(WB)and indirect immunofluorescence(IIF)staining were performed to detect CSFV E2 protein expression in ST cells.Four cDNA libraries were constructed for transcriptome sequencing,and differentially expressed genes(DEGs)were annotated using Gene Ontology(GO)and enriched via Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.DEGs associated with the blood-testis barrier(BTB)were further screened and validated by real-time fluorescence quantitative PCR(qPCR),WB,and IIF.The results showed that E2 protein was expressed in ST cells at both 24 h and 48 h post-CSFV infection.Compared with Control 24 h,a total of 86 significant DEGs(P<0.05)were identified in the CSFV 24 h,and 1481 significant DEGs(P<0.05)were identified in the CSFV 48 h compared with Control 48 h.These DEGs were significantly enriched in 27 GO terms,including biological processes,cellular components,and molecular functions(P<0.01).The most significantly enriched KEGG pathways included TNF signaling,AGE-RAGE signaling,IL-17 signaling,and MAPK signaling pathways(P<0.01).qPCR results showed that,at 24 h,CLDN11,H2AFX,and CDH2 were significantly upregulated(P<0.001 or P<0.01),while TJP1,OCLN,and GJA1 were significantly downregulated(P<0.05).At 48 h,FASLG,TGFB1,IL-6,and H2AFX were significantly upregulated(P<0.001 or P<0.01),whereas TJP1,OCLN,GJA1,CLDN11,and CDH2 were significantly downregulated(P<0.01).WB validation showed that protein levels of TJP1,CLDN11,and GJA1 were significantly downregulated at 24 h(P<0.001 or P<0.01),with CLDN11 showing significant upregulation(P<0.001).At 48 h,TJP1,CLDN11,OCLN,and GJA1 protein levels were all significantly downregulated(P<0.001 or P<0.01).IIF results at 48 h showed that the relative fluorescence area of TJP1 was significantly decreased(P<0.01),and CLDN11,OCLN,and GJA1 were also significantly reduced(P<0.05).These findings were consistent with transcriptome sequencing results,indicating that CSFV infection at both 24 h and 48 h alters the expression of barrier function-related genes in ST cells.This study provides data support for further exploration of how CSFV affects the barrier function of ST cells.