【作者】 程文；

【导师】 余平；

【作者基本信息】 中南大学 ， 抗感染免疫， 2008， 博士

【摘要】 研究目的沙眼衣原体（Chlamydia trachomatis,C.trachomatis）感染除导致沙眼外,也可以引起性传播疾病,导致宫颈炎、输卵管炎、盆腔炎等,最终可致流产、异位妊娠、不孕等严重后遗症。目前认为C.trachomatis感染引起的炎症反应是其致病的主要原因,但确切机制尚不明确,本课题从①C.trachomatis感染引起IL-1α、IL-6、IL-8等炎症因子产生的分子机制;②IL-1α在C.trachomatis诱导IL-8产生中的作用;以及③caspase-1在宿主抵抗衣原体感染和衣原体导致病理损伤中的作用,三个方面探索C.trachomatis感染诱导炎症的机制。为深入研究C.trachomatis致病机制和机体的抗感染免疫反应提供实验依据。研究方法1.用ELISA、RT-PCR方法检测C.trachomatis感染诱导上皮细胞中IL-1α、IL-1β、IL-6、IL-8等细胞因子的产生;用免疫荧光技术观察C.trachomatis感染后细胞内IL-8的表达和亚细胞定位。2.用免疫印迹法检测C.trachomatis感染后ERK、P38、JNK三条MAPK信号通路的激活,用化学抑制剂分别抑制三条通路的活化,观察各条通路在C.trachomatis诱导IL-1α、IL-6、IL-8等细胞因子中的作用。3.用IL-1αsiRNA、IL-1α^-/-鼠巨噬细胞感染实验以及对IL-1α不同表达能力的细胞系感染实验,观察IL-1α在C.trachomatis诱导IL-8中的作用。4.通过IL-1Ra和IL-1α中和性抗体阻碍IL-1RⅠ受体途径,用IL-1RⅠ^-/-鼠巨噬细胞感染实验,观察IL-1α是否通过IL-1RⅠ途径参与C.trachomatis诱导IL-8;并通过免疫荧光观察IL-1α和IL-8亚细胞定位以及真核细胞pDsRed-pro-IL-1α质粒转染实验,进一步明确IL-1α参与C.trachomatis诱导IL-8的机制。5.通过免疫印迹检测C.trachomatis和C.muridarum（小鼠生物型C.trachomatis）感染上皮细胞后caspsase-1的活化;用caspsase-1^-/-鼠生殖道感染C.muridarum,制备C.muridarum生殖道感染动物模型,监测感染鼠IFU情况,观察小鼠对C.muridarum初次感染、再次感染的易感性和C.muridarum的清除,以及初次感染、再次感染后小鼠生殖道病理改变。研究结果1.通过ELISA、RT-PCR方法检测发现C.trachomatis感染可诱导上皮细胞产生IL-1α、IL-1β、IL-6、IL-8等细胞因子,呈时间-剂量依赖性;并且IL-1β、IL-6、IL-8在细胞内和细胞外表达改变的情况是一致的,IL-1α则不同,C.trachomatis感染36h后观察到细胞内IL-1α升高,直至感染60h才检测到细胞外IL-1α;用免疫荧光技术观察C.trachomatis感染的细胞内IL-8表达增高,聚集在高尔基体内。2.用免疫印迹法检测到C.trachomatis感染后可以激活MAPK通路中ERK和P38通路,呈时间依赖性,与C.trachomatis感染诱导IL-1α、IL-1β、IL-6、IL-8等细胞因子的产生具有一定的相关性;通过化学抑制剂分别抑制MAPK三条通路,发现P38信号通路参与C.trachomatis诱导IL-1α、IL-6、IL-8,ERK信号通路仅参与C.trachomatis诱导IL-8。3.通过RT-PCR和ELISA方法发现HT29、HGF两株细胞系不表达内源性IL-1α,C.trachomatis感染也不能诱导HT29、HGF产生IL-8;SiHa、Hela229、Hep2细胞系表达内源性IL-1α,C.trachomatis感染诱导其产生IL-8;IL-1αsiRNA和IL-1α^-/-鼠巨噬细胞感染实验进一步证实C.trachomatis通过IL-1α诱导IL-8产生。4.通过IL-1Ra和IL-1α中和抗体阻碍IL-1α与IL-1RⅠ结合,发现在C.trachomatis感染48h,阻断IL-1RⅠ受体途径并不影响IL-8产生,C.trachomatis感染72h阻断IL-1RⅠ受体途径可以部分显著地抑制IL-8产生。IL-1RⅠ^-/-鼠巨噬细胞C.trachomatis感染实验,显示在C.trachomatis感染48h,IL-1RⅠ^-/-鼠巨噬细胞IL-8产生与野生对照鼠无差异。免疫荧光观察发现C.trachomatis刺激细胞内IL-1α进入细胞核,同时诱导IL-8产生;将pDsRed-pro-IL-1α质粒转染Hela229细胞发现,pDsRed-pro-IL-1α可以进入细胞核诱导IL-8产生。5.免疫印迹实验检测到C.trachomatis和C.muridarum感染后可活化caspase-1;将C.muridarum感染caspase-1^-/-鼠生殖道,制备C.muridarum生殖道感染动物模型,发现caspase-1不影响小鼠对C.muridarum的易感性,无论初次还再次感染,caspase-1^-/-鼠清除C.muridarum能力与对照组相比无显著差别。制备病理切片评估小鼠生殖道炎症程度,显示caspase-1^-/-鼠输卵管的炎症损伤在初次感染后较野生对照组显著减轻（p＜0.05）。结论1.活化MAPK/ERK通路参与C.trachomatis诱导炎症因子IL-8的产生,活化MAPK/P38通路参与C.trachomatis诱导炎症因子IL-1α、IL-6、IL-8的产生。2.细胞内IL-1α可以通过一条非IL-1RⅠ依赖途径介导C.trachomatis诱导IL-8产生。3.Caspase-1参与C.muridarum对小鼠上生殖道的病理损伤,而不影响小鼠抵抗C.muridarum感染。更多还原

【Abstract】 ObjectiveInfection of Chlamydia trachomatis can lead to not only trachomatis but also sexually transmitted diseases such as cervicitis,salpingitis,pelvic inflammatory disease,subsequent tubal infertility,ectopic pregnancy, abortion et al.The inflammation induced by Chlamydia is contributed to the diseases,but the definite mechanism is unclear.In this report:①The signaling pathway mechanisms of chlamydial induction of proinflammatory factors including IL-1α,IL-6,IL-8 was explored.②The effects of IL-1αon chlamydial induction of IL-8 was studied.③The effects of caspase-1 on the resist to chlamydial infection and on the upper genital tract pathologies caused by Chlamydia were also studied.From all these the mechanisms of Chlamydia-induced diseases are tried to be found and these studies can establish the experimental foundation for therapy and prevention of Chlamydia.Methods1.To identify the production of proinflammatory cytokines such as IL-1α,IL-1β,IL-6,IL-8 et al induced by Chlamydia via ELISA and RT-PCR.To identify the production and location of intracellular IL-8 induced by Chlamydia via immunofluorescence assay.2.To identify the activation of different MAPK pathways（ERK or P38 or JNK）induced by Chlamydia via western-blot.And to identify the exact pathway responsible for the induction of different inflammatory cytokines such as IL-1α,IL-6 and IL-8 et al by using the chemical inhibitors for different signaling pathways via western-blot.3.To identify the role of IL-1αin Chlamydia-induced IL-8 via the methods of IL-1αsiRNA,infection experiment of IL-1αgene "knockout mice macrophage and infection experiment of different cell line which express different level of IL-1α.4.To identify the way that IL-1αparticipates in the Chlarnydia-induced IL-8 via the methods of IL-1Ra and IL-1αneutralization antibody blocking of the IL-1R I pathway,infection experiment of IL-1RⅠ^-/- mice macrophage.And to identify the way that IL-1αcontribute to the production of IL-8,localization of intracellular IL-1αthrough chlamydial infection experiment and transfection of pDsRed-pro-IL-1αplasmid into mammalian cells via immunoflourescence assay.5.To identify the activation of caspase-1 induced by Chlamydia via western-blot.Establishing the mouse genital C.muridarum infection model in order to observe the role of caspase-1 on the susceptibility to Chlamydia and on the ability of bacteria clearance between caspase-1^-/-mice and wild type through monitoring the genital shedding after chlamydial infection.And to assess the genital inflammation damage through pathological studies after primary and secondary C.muridarum infection.Result1.Chlamydia can induce inflammatory cytokines such as IL-1α, IL-1β,IL-6,IL-8 and is time-responsible.The production of extracellular IL-6,IL-8 is identical with the intracellular ones,while the extracellular IL-1αis 24 hours delayed after the detection of intracellular IL-1α.And Chlamydia-induced IL-8 is co-localized with Golgi via immunoflourescence.2.Chlamydia can induce the activation of MAPK/ERK and MAPK/P38 signaling pathways,and is time-responsible.The activation of MAPK/ERK and MAPK/P38 pathways is associated with the production of the proinflammmatory cytokines.Inhibiting different MAPK pathways by the chemical inhibitors,showed that MAPK/ERK pathway is contribute to the Chlamydia-induced IL-8 and MAPK/P38 pathway is contribute to the Chlamydia-induced IL-1α,IL-6 and IL-8.3.It showed that the IL-1αmRNA level and protein expression can not be detected in HT29 and HGF cell lines even after chlamydial infection.Compared with the cell lines such as SiHa、Hela229 and Hep2 which can express IL-1α,IL-8 can be detected by neither of these tow cell lines.To further study the role of IL-1αin Chlamydiainduced IL-8,experiments of IL-1αsiRNA and IL-1α^-/-mice macrophage infection were done.The studies showed that Chlamydia-induced IL-8 is dependent on IL-1α.4.Blocking of the IL-1RⅠpathway by IL-1Ra and IL-1αneutralization antibody showed that Chlamydia-induced IL-8 is not dependent on the IL-1RⅠpathway at 48h post infection,while is partially dependent on the IL-1RⅠpathway at 72h post infection.The studies showed that there is no significant difference between the production of IL-8 from Chlymadia-infected macrophage of IL-1RⅠ^-/-mice and of wild type mice.It also showed that Chlamydia can stimulate intracellular IL-1αto translocate into nuclear and IL-8 staining was found in Chlamydia-infected cells via immunoflourescence assay.And transfecting pDsRed-IL-1αinto mammalian cells showed that the fusion protein can move into nuclear and induce IL-8 production compared with the empty plasmid.5.Chlamydia trachomatis can activate caspsase-1 and processing IL-1β.Infection of the mouse genital tract with C.muridarum,showed that caspsae-1 had no effect on the susceptibility to Chlamydia and had no effect on the bacteria clearance through monitoring the genital shedding.The studies also showed that caspase-1 can significantly enhance the oviduct inflammation damages compared with the wild type mice（p＜0.05）.Conclusion1.Activation of MAPK/ERK pathway is essential for the production of IL-8 induced by Chlamydia trchomatis,activation of MAPK/P38 pathway is essential for the production of IL-1α,IL-6 and IL-8 induced by Chlarnydia trchomatis.2.Intracellular interleukin-1αmediates interleukin-8 production induced by Chlamydia trachomatis infection via a mechanism independent of typeⅠinterleukin-1 receptor.3.Caspas-1 contributes to Chlamydia-induced upper urogenital tract inflammatory pathologies,but has no effect on the susceptibility to Chlamydia and on the ability of bacteria clearance.更多还原