大肠杆菌对奶牛子宫内膜组织合成分泌IL-1β、IL-6和PGE_2的影响
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摘要
基于奶牛子宫内膜组织体外培养方法,分别采用浓度为1×10~5,1×10~6 CFU/mL的致病性大肠杆菌处理体外培养的子宫内膜组织12,24 h后用HE染色方法评价组织损伤情况,ELISA法检测组织培养液上清中IL-1β,IL-6和PGE_2的分泌变化,RT-qPCR法检测IL-1β和IL-6基因表达变化。结果显示:与对照组相比,大肠杆菌分别处理12,24 h后奶牛子宫内膜组织中子宫内膜上皮细胞和腺上皮细胞脱落、坏死、崩解,并且显著提高奶牛子宫内膜组织IL-1β,IL-6和PGE_2的分泌及IL-1β和IL-6的mRNA的表达量。结果表明:成功建立了体外大肠杆菌型奶牛子宫内膜炎模型,且IL-1β,IL-6和PGE_2在大肠杆菌引起的奶牛子宫内膜组织炎症反应中发挥着重要的调控作用。
Endometrial explants in vitro culture,were stimulated by endometrial pathogenic E.coli(1×10~5,1×10~6 CFU/mL) for 12,24 h,followed by the detection of endometrial explants damage by HE,the section of IL-1β,IL-6 and PGE_2 in the endometrial explants supernatant by ELISA and the differential expression of IL-1β and IL-6 by RT-PCR.The results showed that the histological sections of E.coli induced endometrium indicated that the endometrial glands were not clearly visible,and endometrial epithelial cells and glandular epithelial cells had disappeared after treatment of E.coli for 12,24 h,and the IL-1β,IL-6 and PGE_2 section and IL-1β,IL-6 mRNA expression were significantly up-regulated compared with control group.The results indicated that the in vitro cultivation method of Escherichia coli induced bovine endometrial tissue was successfully established,and IL-1β,IL-6 and PGE_2 may play an important role in the inflammatory response of bovine endometrium induce by E.coli.
Endometrial explants in vitro culture,were stimulated by endometrial pathogenic E.coli(1×10~5,1×10~6 CFU/mL) for 12,24 h,followed by the detection of endometrial explants damage by HE,the section of IL-1β,IL-6 and PGE_2 in the endometrial explants supernatant by ELISA and the differential expression of IL-1β and IL-6 by RT-PCR.The results showed that the histological sections of E.coli induced endometrium indicated that the endometrial glands were not clearly visible,and endometrial epithelial cells and glandular epithelial cells had disappeared after treatment of E.coli for 12,24 h,and the IL-1β,IL-6 and PGE_2 section and IL-1β,IL-6 mRNA expression were significantly up-regulated compared with control group.The results indicated that the in vitro cultivation method of Escherichia coli induced bovine endometrial tissue was successfully established,and IL-1β,IL-6 and PGE_2 may play an important role in the inflammatory response of bovine endometrium induce by E.coli.
引文
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[24] SHELDON I M,CRONIN J G,HEALEY G D,et al.Innate immunity and inflammation of the bovine female reproductive tract in health and disease[J].Reproduction,2014,148(3):41-51.
[25] 张楠,张莹,曹金山,等.EP2受体和FP受体对奶牛输卵管上皮细胞输卵管蛋白的影响[J].中国兽医学报,2017,37(7):1394-1399.
[26] LYU A,CHEN J J,WANG H C,et al.Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury[J].J Zhejiang Univ Sci B,2017,18(6):481-491.
[27] HUANG B,XIAO D,TAN B,et al.Chitosan oligosaccharide reduces intestinal inflammation that involves calcium-sensing receptor (CaSR) activation in lipopolysaccharide (LPS)-challenged piglets[J].J Agric Food Chem,2016,64(1):245-252.
[28] CHAPWANYA A,MEAE K G,DOHER T Y,et al.Endometrial epithelial cells are potent producers of tracheal antimicrobial peptide and serum amyloid A3 gene expression in response to E.coli stimulation[J].Vet Immunol Immunopathol,2013,151:157-162.
[29] ZHANG S,LIU B,MAO W,et al.The effect of prostaglandin E2 receptor (PTGER2) activation on growth factor expression and cell proliferation in bovine endometrial explants,Prostaglandins[J].Prostaglandins Leukot Essent Fatty Acids,2017,122:16-23.
[30] NISHIMURA T,ZHAO X,GAN H,et al.The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis[J].FASEB J,2013,B27(9):3827-3836.
[31] WACLAWIK A,JABBOUR H N,BLITEK A,et al.Estradiol-17beta,prostaglandin E2 (PGE2),and the PGE2 receptor are involved in PGE2 positive feedback loop in the porcine endometrium[J].Endocrinology,2009,150(8):3823-3832.
[32] LARA E,VELASQUEZ A,CABEZAS J,et al.Endometritis and in vitro PGE2 challenge modify properties of cattle endometrial mesenchymal stem cells and their transcriptomic profile[J].Stem Cells Int,2017,2017:4297639.
[2] GILBERT R O,SHIN S T,GUARD C L,et al.Prevalence of endometritis and its effects on reproductive performance of dairy cows[J].Theriogenology,2005,64(9):1879.
[3] 何光祥,李加泽.如何提高奶牛繁殖力[J].安徽农学通报,2010,16(2):35.
[4] 王军,曹梅艳,姜才,等.内蒙古局部地区奶牛不孕症的调查及子宫内膜炎性不孕症奶牛子宫内细菌分离鉴定[J].中国奶牛,2008(10):43-46.
[5] 李宗波,付才,孔德江.奶牛子宫内膜炎研究进展[J].中国乳业,2010(2):62-65.
[6] LE BLANCE S J.Reproductive tract inflammatory disease in postpartum dairy cows[J].Animal,2014,8 (Suppl) 1:54-63.
[7] HERATH S,LILLY S T,SANTOS N R,et al.Expression of genes associated with immunity in the endometrium of cattle with disparate postpartum uterine disease and fertility[J].Reprod Biol Endocrinol,2009(7):55.
[8] PECCHI E,DALLAPORTA M,JEAN A,et al.Prostaglandins and sickness behavior:old story,new insights[J].Physiol Behavior,2009,97(3/4):279-292.
[9] JOHNSON H,TORRES C G,CARVALLO F,et al.Expression of selected transcripts in postpartum of primiparous Holstein cows with clinical and subclinical endometritis[J].Anim Reprod Sci,2015,156:34-39.
[10] ZHANG S,LIU B,MAO W,et al.The effect of prostaglandin E2 receptor (PTGER2) activation on growth factor expression and cell proliferation in bovine endometrial explants[J].Prostaglandins Leukot Essent Fatty Acids,2017,122:16-23.
[11] ZHANG S,LIU B,MAO W,et al.F2α-PTGFRsignalling activation,growth factor expression and cell proliferation in bovine endometrial explants[J].Reprod Fertil Dev,2017,29:2195-2205.
[12] 李华.平板菌落计数的改进方法[J].生物学通报,2006,41(1):51.
[13] 李二卫.食品卫生微生物学检验菌落总数测定方法的探讨[J].中国卫生检验杂志,2010(8):1940-1941.
[14] AMOS M R,HEALEY G D,GOLDSTONE R J,et al.Differential endometrial cell sensitivity to a cholesterol-dependentcytolysin links Trueperella pyogenes to uterine disease in cattle[J].Biol Reprod,2014,90:54.
[15] SHELDON I M,CRONIN J,GOETZE L,et al.Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle[J].Biol Reprod,2009,81:1025-1032.
[16] DONFRIO G,RAVANETTI L,CAVIRANI S,et al.Bacterial infection of endometrial stromal cells influences bovine herpesvirus 4 immediate early gene activation:a new insight into bacterial and viral interaction for uterine disease[J].Reproduction,2008,136:361-366.
[17] 苗淑媛,刘中柱,王洪武,等.前列腺素类药物在兽医临床上的应用[J].养殖技术顾问,2013(12):176.
[18] WILLIAMS E J,FISCHER D P,NOAKES D,et al.The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow[J].Theriogenology,2007,68:549-559.
[19] CASTRO L V,ESPEJEL N A,BARROSO G,et al.Preserved ex vivo inflammatory status in decidual cells from women with preterm labor and subclinical intrauterine infection[J].PLoS One,2012,7(8):e43605.
[20] LEBLANC S J.Interactions of metabolism,inflammation,and reproductive tract health in the postpartum period in dairy cattle[J].Reprod Domestic Anim,2012,47(5):18-30.
[21] STICHTENOTH D O,THOREN S,BIAN H,et al.Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells[J].J Immunol,2001,167:469-474.
[22] THORéN S,JOHAN P J,Coordinate up- and down-regulation of glutathione-dependent prostaglandin E synthase cyclooxygenase-2 in A549 cells inhibition by NS398 leukotriene C4[J].Eur J Biochem,2000,267:6428-6434.
[23] HERATH S,LILLY S T,FISCHER D P,et al.Bacterial lipopolysaccharide induces an endocrine switch from prostaglandin F2alpha to prostaglandin E2 in bovine endometrium[J].Endocrinology,2009,150(4):1912-1920.
[24] SHELDON I M,CRONIN J G,HEALEY G D,et al.Innate immunity and inflammation of the bovine female reproductive tract in health and disease[J].Reproduction,2014,148(3):41-51.
[25] 张楠,张莹,曹金山,等.EP2受体和FP受体对奶牛输卵管上皮细胞输卵管蛋白的影响[J].中国兽医学报,2017,37(7):1394-1399.
[26] LYU A,CHEN J J,WANG H C,et al.Punicalagin protects bovine endometrial epithelial cells against lipopolysaccharide-induced inflammatory injury[J].J Zhejiang Univ Sci B,2017,18(6):481-491.
[27] HUANG B,XIAO D,TAN B,et al.Chitosan oligosaccharide reduces intestinal inflammation that involves calcium-sensing receptor (CaSR) activation in lipopolysaccharide (LPS)-challenged piglets[J].J Agric Food Chem,2016,64(1):245-252.
[28] CHAPWANYA A,MEAE K G,DOHER T Y,et al.Endometrial epithelial cells are potent producers of tracheal antimicrobial peptide and serum amyloid A3 gene expression in response to E.coli stimulation[J].Vet Immunol Immunopathol,2013,151:157-162.
[29] ZHANG S,LIU B,MAO W,et al.The effect of prostaglandin E2 receptor (PTGER2) activation on growth factor expression and cell proliferation in bovine endometrial explants,Prostaglandins[J].Prostaglandins Leukot Essent Fatty Acids,2017,122:16-23.
[30] NISHIMURA T,ZHAO X,GAN H,et al.The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis[J].FASEB J,2013,B27(9):3827-3836.
[31] WACLAWIK A,JABBOUR H N,BLITEK A,et al.Estradiol-17beta,prostaglandin E2 (PGE2),and the PGE2 receptor are involved in PGE2 positive feedback loop in the porcine endometrium[J].Endocrinology,2009,150(8):3823-3832.
[32] LARA E,VELASQUEZ A,CABEZAS J,et al.Endometritis and in vitro PGE2 challenge modify properties of cattle endometrial mesenchymal stem cells and their transcriptomic profile[J].Stem Cells Int,2017,2017:4297639.
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