猪圆环病毒2型感染对猪小肠上皮细胞Claudin-1蛋白和闭锁小带蛋白-1基因mRNA表达的影响
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摘要
紧密连接在维持肠道黏膜完整性和免疫系统正常发挥功能中作用重大。【目的】旨在探讨猪圆环病毒2型(PCV2)感染对猪小肠上皮细胞(IPEC)紧密连接的影响。【方法】利用基因克隆获得Claudin-1蛋白和闭锁小带蛋白-1 (ZO-1)基因的重组质粒并建立荧光定量RT-PCR标准曲线;利用所建立的方法检测PCV2感染的IPEC中Claudin-1蛋白和闭锁小带蛋白-1mRNA的动态变化。【结果】构建的两种紧密连接蛋白基因的重组质粒荧光定量RT-PCR标准曲线线性关系良好,相关系数均达0.99以上。PCV2感染的IPEC中Claudin-1蛋白和ZO-1mRNA表达在4、12、24、48、72h均显著下降。【结论】PCV2感染可抑制猪小肠上皮细胞紧密连接蛋白相关基因的表达,破坏细胞间的紧密连接,增加肠道通透性。
Tight junctions play an important role in maintaining the integrity of intestinal mucosa and the normal functioning of the immune system.【Objective】The aim of this study was to investigate the effect of PCV2 infection on tight junction of intestinal porcine epithelial cells(IPEC).【Methods】the recombinant plasmids of Claudin-1 and Zonula Occluden-1(ZO-1)gene were obtained by gene cloning and their standard curves of fluorescence quantitative(FQ)RT-PCR were established.The dynamic changes of Claudin-1 and ZO-1 mRNA in IPEC infected with PCV2 were detected by the method established.【Results】The standard curves of the recombinant plasmid(FQ)RT-PCR of the two tight junction proteins were linear,and their correlation coefficients were above 0.99.The expression of Claudin-1 and ZO-1 mRNA in PCV2 infected IPEC decreased significantly at 4,12,24,48 and 72 hafter infection.【Conclusion】PCV2 infection can inhibit the expression of tight junction protein related genes in IPECs,which maybe destroy the tight junctions between cells,and increase intestinal permeability.
Tight junctions play an important role in maintaining the integrity of intestinal mucosa and the normal functioning of the immune system.【Objective】The aim of this study was to investigate the effect of PCV2 infection on tight junction of intestinal porcine epithelial cells(IPEC).【Methods】the recombinant plasmids of Claudin-1 and Zonula Occluden-1(ZO-1)gene were obtained by gene cloning and their standard curves of fluorescence quantitative(FQ)RT-PCR were established.The dynamic changes of Claudin-1 and ZO-1 mRNA in IPEC infected with PCV2 were detected by the method established.【Results】The standard curves of the recombinant plasmid(FQ)RT-PCR of the two tight junction proteins were linear,and their correlation coefficients were above 0.99.The expression of Claudin-1 and ZO-1 mRNA in PCV2 infected IPEC decreased significantly at 4,12,24,48 and 72 hafter infection.【Conclusion】PCV2 infection can inhibit the expression of tight junction protein related genes in IPECs,which maybe destroy the tight junctions between cells,and increase intestinal permeability.
引文
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[12]Morit K,Furuse M,Fujimoto K,Tsukita S.Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96:511-516
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[14]Hollander D,Vadheim CM,Brettholz E,Petersen GM,Delahunty T.Increased intestinal permeability in patients with Crohn’s disease and their relatives.A possible etiologic factor[J].Digestive&Liver Disease,1986,33(8):883-885
[15]Larre I,Ruiz A,Shoshani L.New diseases derived or associated with the tight junction[J].Archives of Medical Research,2007,38:465-478
[16]Fanning AS,Jameson BJ,Jesaitis LA,Anderson JM.The tight junction protein ZO-1establishes a link between the trans membrane protein occludin and the actin cytoskeleton[J].Journal of Biological Chemistry,1998,273(45):29745-29753
[17]Nordmann P,Poirel L.Strategies for identification of carbap-enemase-producing entero bacteriaceae[J].Journal of Antimicrobial Chemotherapy,2013,68(3):487-489
[18]Drago S,El AR,Di PM,Grazia CM,Tripathi A.Gliadin,zonulin and gut permeability:Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines[J].Scandinavian Journal of Gastroenterology,2006,41:408-419
[19]Anderson JM.Molecular structure of tight junctions and their role in epithelial transport[J].News in Physiological Science,2001,16:126-130
[20]Patrick DM,Leone AK,Shellenberger JJ,Dudowicz KA,King JM.Proinflammatory cytokines tumor necrosis factoralpha and interferon-gamma modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling[J].BMC Physiology,2006,21(6):2
[2]王小英,程爱国.肠道黏膜屏障功能及其检测方法研究进展[J].河北联合大学学报:医学版,2009,11(5):653-654
[3]Ma TY.Intestinal epithelial barrier dysfunction in Crohn’s disease[J].Proceedings of the Society for Experimental Biology&Medicine Society for Experimental Biology&Medicine,1997,214(4):318-327
[4]Al-Sadi R,Ye D,Boivin M,Guo S,Hashimi M.Interleukin-6modulation of intestinal epithelial tight junction permeability is mediated by JNK pathway activation of claudin-2gene[J].PLoS One,2014,9(3):e85345
[5]Visser J,Rozing J,Sapone A,Lammers K,Fasano A.Tight junctions,intestinal permeability,and autoimmunity:celiac disease and type 1diabetes paradigms[J].Annals of the New York Academy of Sciences,2009,1165:195-205
[6]韦志坤,程爱国.肠黏膜细胞的紧密连接与肠壁通透性的研究进展[J].世界华人消化杂志,2011(4):394-399
[7]Yan M,Zhu L,Yang Q.Infection of Porcine Circovirus 2(PCV2)in intestinal porcine epithelial cell line(IPEC-J2)and Interaction between PCV2and IPEC-J2 Microfilaments[J].Virology Journal,2014,11(1):193
[8]王丽琼,何乐,李焕荣.PCV2感染猪血管内皮细胞相关免疫调节分子的表达变化[J].北京农学院学报,2013,28(3):33-36
[9]陈景杰,陈新瑶,董星,秦韬,李健,黄一帆.猴头菇多糖对氧化应激状态下猪小肠上皮细胞紧密连接相关基因表达的影响[J].中国兽医科学,2017,47(1):128-134
[10]Yang N,Qiao J,Liu S,Zou Z,Zhu L.Change in the immune function of porcine iliac artery endothelial cells infected with porcine circovirus type 2and its inhibition on monocyte derived dendritic cells maturation[J].Proceedings of the National Academy of Sciences of the United States of America,2017,12(10):e0186775
[11]邹战明.PCV2感染猪肠粘膜免疫功能变化分子基础[D].北京:北京农学院,2016
[12]Morit K,Furuse M,Fujimoto K,Tsukita S.Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96:511-516
[13]秦环龙,高志光.肠上皮细胞紧密连接在肠屏障中的作用研究进展[J].世界华人消化杂志.2005,13(4):443-447
[14]Hollander D,Vadheim CM,Brettholz E,Petersen GM,Delahunty T.Increased intestinal permeability in patients with Crohn’s disease and their relatives.A possible etiologic factor[J].Digestive&Liver Disease,1986,33(8):883-885
[15]Larre I,Ruiz A,Shoshani L.New diseases derived or associated with the tight junction[J].Archives of Medical Research,2007,38:465-478
[16]Fanning AS,Jameson BJ,Jesaitis LA,Anderson JM.The tight junction protein ZO-1establishes a link between the trans membrane protein occludin and the actin cytoskeleton[J].Journal of Biological Chemistry,1998,273(45):29745-29753
[17]Nordmann P,Poirel L.Strategies for identification of carbap-enemase-producing entero bacteriaceae[J].Journal of Antimicrobial Chemotherapy,2013,68(3):487-489
[18]Drago S,El AR,Di PM,Grazia CM,Tripathi A.Gliadin,zonulin and gut permeability:Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines[J].Scandinavian Journal of Gastroenterology,2006,41:408-419
[19]Anderson JM.Molecular structure of tight junctions and their role in epithelial transport[J].News in Physiological Science,2001,16:126-130
[20]Patrick DM,Leone AK,Shellenberger JJ,Dudowicz KA,King JM.Proinflammatory cytokines tumor necrosis factoralpha and interferon-gamma modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling[J].BMC Physiology,2006,21(6):2