CD44通过影响猪流行性腹泻病毒复制调节钠氢交换体3活性  

CD44 Regulates Na^(+)/H^(+)Exchanger 3 Activity by Influencing Porcine Epidemic Diarrhea Virus Replication

作　　者：王静[1] 张淑娟 胡霞 刘向阳 张兴翠[1] 宋振辉 WANG Jing;ZHANG Shujuan;HU Xia;LIU Xiangyang;ZHANG Xingcui;SONG Zhenhui(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;College of Animal Medicine,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]西南大学动物医学院,重庆402460 [2]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《畜牧兽医学报》2024年第5期2176-2185,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：中央高校基本业务基金资助项目(XDJK2020RC001);重庆市研究生科研创新项目(CYS19137)。

摘　　要：本研究旨在研究CD44(cluster of differentiation 44)对感染猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)的猪小肠上皮细胞(IPEC-J2)中钠氢交换体3(NHE3)表达及膜转移的影响。以IPEC-J2为细胞模型,采用RT-qPCR和Western blot检测感染PEDV后不同时间点IPEC-J2细胞中NHE3和PEDV N表达量变化;转染质粒调控IPEC-J2中CD44表达后,采用TCID50和Western blot检测PEDV感染后不同时间点PEDV复制水平和NHE3蛋白表达变化,采用火焰原子吸收法检测IPEC-J2细胞内外Na^(+)浓度变化。转录组数据和细胞试验结果显示,与对照组相比,PEDV感染后IPEC-J2细胞中CD44蛋白表达水平和mRNA表达量均呈上调趋势,24~48 h内上升显著(P<0.05),而PEDV N蛋白表达水平在12~48 h内则呈显著下降趋势(P<0.05)。此外,CD44重组质粒转染试验结果显示,与PEDV感染组相比,过表达CD44后感染PEDV组细胞中病毒滴度和PEDV N蛋白表达水平显著降低(P<0.05),而干扰CD44后感染PEDV组细胞中病毒滴度和PEDV N蛋白表达水平则显著上升(P<0.05)。以上结果表明,高表达CD44具有抑制PEDV复制的作用,干扰CD44后PEDV复制增多。同时,为研究PEDV感染情况下,CD44是否参与了IPEC-J2细胞中NHE3表达的调节,采用Western blot和火焰原子吸收法检测了调节CD44后膜NHE3蛋白的表达水平和细胞内外Na^(+)浓度。结果表明,过表达CD44显著促进了膜NHE3蛋白的表达和活性(P<0.05),细胞内外Na^(+)浓度逐渐恢复正常水平。相反,干扰CD44显著降低了膜NHE3蛋白的表达和活性(P<0.05),细胞内外Na^(+)浓度呈现失衡状态。结果提示,CD44可能是缓解PEDV引发仔猪腹泻的潜在治疗靶点,它通过抑制IPEC-J2细胞中PEDV的复制来增加转移至质膜上的NHE3数量,从而维持细胞内外Na^(+)运转平衡。This study aimed to investigate the effect of CD44(cluster of differentiation 44)on the expression and membrane transfer of Na^(+)/H^(+)exchanger 3(NHE3)in porcine intestinal epithelial cells(IPEC-J2)infected with porcine epidemic diarrhea virus(PEDV).Using IPEC-J2 as the cell model,the expression of CD44 in IPEC-J2 was regulated by transfecting plasmids,the expression of PEDV N and NHE3 in IPEC-J2 cells at different time points after infection with PEDV was detected by RT-qPCR and Western blot,and the changes of Na^(+)concentration inside and outside IPEC-J2 cells were detected by flame atomic absorption spectrometry.Using IPEC-J2 as the cell model,the expression of NHE3 and PEDV N in IPEC-J2 cells at different time points after infection with PEDV was detected by RT-qPCR and Western blot.After the transfection plasmid regulated the expression of CD44 in IPEC-J2,TCID 50 and Western blot were used to detect the changes of PEDV replication level and NHE3 protein expression at different time points after PEDV infection,and the changes of Na^(+)concentration inside and outside IPEC-J2 cells were detected by flame atomic absorption method.Transcriptome data and cell experimental results showed that compared with the control group,the expression level of CD44 protein and mRNA expression in IPEC-J2 cells after PEDV infection showed an upward trend,and increased significantly within 24-48 h(P<0.05),while the expression level of PEDV N protein decreased significantly within 12-48 h(P<0.05).In addition,the results of CD44 recombinant plasmid transfection experiments showed that the viral titer and PEDV N protein expression levels in cells infected with PEDV group after overexpression of CD44 were significantly reduced(P<0.05),while the viral titer and PEDV N protein expression levels in cells infected with PEDV group after interference with CD44 were significantly increased(P<0.05).These results showed that overexpression of CD44 had the effect of inhibiting PEDV replication,and PEDV replication increased after interferin

关 键 词：CD44 猪流行性腹泻病毒 钠氢交换体3 钠离子 

分 类 号：S852.659.6[农业科学—基础兽医学]