抗菌肽LL-37诱导大鼠膀胱壁肥大细胞的炎症反应
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摘要
目的在实验室中构建抗菌肽LL-37大鼠膀胱灌注动物模型,研究该模型条件下实验动物膀胱壁肥大细胞的炎症反应。方法取8～12周龄雌性SD大鼠作为实验动物,以320μM抗菌肽LL-37溶液灌注大鼠膀胱建立实验动物模型。建模完成后,通过HE染色观察大鼠膀胱壁组织形态改变,透射电镜观察大鼠膀胱壁肥大细胞脱颗粒现象。采用ELISA试剂盒检测大鼠尿液和膀胱壁组织标本中过氧化物酶(MPO)的含量,采用免疫酶标染色观察单核细胞趋化蛋白1 (MCP1)、糜蛋白酶、类胰蛋白酶在大鼠膀胱壁中的表达和分布。结果与对照组相比,实验组大鼠膀胱壁出现显著的炎症水肿反应;透射电镜观察可见,实验动物膀胱壁肥大细胞出现明显脱颗粒现象。ELISA数据显示,相较对照组,MPO在实验组尿液及膀胱壁组织标本中均有较高含量(P<0.001)。免疫酶标染色结果显示,实验组大鼠膀胱壁组织中MCP1 (P=0.000 6)、糜蛋白酶(P<0.000 1)和类胰蛋白酶(P<0.000 1)的表达均显著上调。结论抗菌肽LL-37可诱导实验大鼠膀胱壁发生炎症反应,其中肥大细胞脱颗粒现象显著,相关炎症因子表达明显上调。
Objective To establish an in vivo female rat model of bladder perfusing antibacterial peptide LL-37, and to study the inflammatory reaction of bladder wall mast cells under the condition. Me thods 8 to 12 weeks old female SD rats were used as experimental animals; the experimentalmodel was established by perfusing the rat bladder with 320 μM LL-37 solution.The morphology of rat bladder wall was observed by HE stains; the degranulation of mast cells in rat bladder was observed by transmission electron microscopy.The content of mast cell peroxidase(MPO) in rat urine and rat bladder wall was surveyedby ELISA kit.The expression of monocyte chemoattractant protein 1(MCP1), mast cell chymotrypsin and mast cell tryptase in rat bladder were examinedby Immunohistochemistry(IHC).Re s ults Compared with control group, the experimental group ratshad significant inflammation and edema reaction in the bladder wall;and an obviousmast celldegranulationwas observed in the bladder wall specimen of the experimental group ratsby transmission electron microscopy.The ELISA data showed that MPO had a higher content in the urine and bladder wall tissues in the experimental group(P < 0.001).The IHC showed that the expression of MCP1(P = 0.0006),chymotrypsin(P < 0.0001) and tryptase(P <0.0001) in the rat bladder of experimental group were significantly up-regulated.Conclus ions The antibacterial peptide LL-37 can induce inflammatory reaction in the bladder wall of experimental rats,in which the degranulation of mast cells is significant,and the expression of mast cell related inflammatory factors is up-regulated.
Objective To establish an in vivo female rat model of bladder perfusing antibacterial peptide LL-37, and to study the inflammatory reaction of bladder wall mast cells under the condition. Me thods 8 to 12 weeks old female SD rats were used as experimental animals; the experimentalmodel was established by perfusing the rat bladder with 320 μM LL-37 solution.The morphology of rat bladder wall was observed by HE stains; the degranulation of mast cells in rat bladder was observed by transmission electron microscopy.The content of mast cell peroxidase(MPO) in rat urine and rat bladder wall was surveyedby ELISA kit.The expression of monocyte chemoattractant protein 1(MCP1), mast cell chymotrypsin and mast cell tryptase in rat bladder were examinedby Immunohistochemistry(IHC).Re s ults Compared with control group, the experimental group ratshad significant inflammation and edema reaction in the bladder wall;and an obviousmast celldegranulationwas observed in the bladder wall specimen of the experimental group ratsby transmission electron microscopy.The ELISA data showed that MPO had a higher content in the urine and bladder wall tissues in the experimental group(P < 0.001).The IHC showed that the expression of MCP1(P = 0.0006),chymotrypsin(P < 0.0001) and tryptase(P <0.0001) in the rat bladder of experimental group were significantly up-regulated.Conclus ions The antibacterial peptide LL-37 can induce inflammatory reaction in the bladder wall of experimental rats,in which the degranulation of mast cells is significant,and the expression of mast cell related inflammatory factors is up-regulated.
引文
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[2] VAN DE MERWE J P,NORDLING J,BOUCHELOUCHE P,et al. Diagnostic criteria,classification,and nomenclature for painful bladder syndrome/interstitial cystitis:an ESSIC proposal[J].Eur Urol,2008,53(1):60-67.
[3] HANNO P,DMOCHOWSKI R. Status of international consensus on interstitial cystitis/bladder pain syndrome/painful bladder syndrome:2008 snapshot[J].Neurourol Urodyn,2009,28(4):274-286.
[4] THEOHARIDES T C,KEMPURAJ D,SANT G R. Mast cell involvement in interstitial cystitis:a review of human and experimental evidence[J].Urology,2001,57(6 Suppl1):47-55.
[5] HURST R E,MOLDWIN R M,MULHOLLAND S G.Bladder defense molecules,urothelial differentiation[J].urinary biomarkers,and interstitial cystitis[J].Urology,2007,69(4 Suppl):17-23.
[6] NIJNIK A,HANCOCK R E. The roles of cathelicidin LL-37 in immune defences and novel clinical applications[J].Curr Opin Hematol,2009,16(1):41-47.
[7] CHROMEK M,SLAMOVA Z,BERGMAN P,et al. The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection[J].Nat Med,2006,12(6):636-641.
[8] JOHANSSON J,GUDMUNDSSON G H,ROTTENBERG ME,et al. Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37[J].Journal of Biological Chemistry,1998,273(6):3718-3724.
[9] OOTTAMASATHIEN S,JIA W,MCCOARD L,et al. A murine model of inflammatory bladder disease:cathelicidin peptide induced bladder inflammation and treatment with sulfated polysaccharides[J].J Urol,2011,186(4 Suppl):1684-1692.
[10] OOTTAMASATHIEN S,JIA W,ROUNDY L M,et al.Physiological relevance of LL-37 induced bladder inflammation and mast cells[J].J Urol,2013,190(4 Suppl):1596-1602.