【作者】 董亚珞；

【导师】 刘沛；

【作者基本信息】 中国医科大学 ， 内科学， 2008， 博士

【摘要】 前言肠黏膜屏障是机体最重要的免疫防御屏障,将机体与肠道内的外源性物质隔离开来,避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障包括肠上皮细胞屏障、免疫屏障和微生物屏障,肠上皮细胞屏障是最重要的一道屏障,是肠黏膜屏障具有选择性通透的基础。目前研究发现多种疾病的发生（如炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等）都涉及到肠上皮细胞屏障通透性增高和细菌的定位转移,并且在这些情况下,往往伴有细胞因子的参与,比较常见的血清肿瘤坏死因子α（TNFα）明显升高,与肠上皮细胞屏障的损害程度呈正相关,抗TNFα抗体可以恢复受损的肠上皮细胞屏障。因此认为TNFα是增加肠上皮细胞屏障通透性的重要因素,但TNFα是否也引起细菌定位转移增加,是否是肠上皮细胞屏障通透性增加的结果,尚有待证实。虽然目前已经形成共识:TNFα可以增加肠上皮细胞屏障的通透性,但TNFα的具体作用机制和方式还存在争议。早期认为与TNFα引起的细胞凋亡密切相关,近年来则倾向于TNFα引起肠上皮细胞屏障损伤是非凋亡依赖性的,可能与PKC、NF-kappaB（nuclear factor-kappaB）、MLCK、MAPK等信号途径有关。大部分有核细胞的表面都有TNF受体,TNFRⅠ和TNFRⅡ介导TNFα所引起的生物效应各有不同,TNFα究竟是通过哪一种受体引起肠上皮细胞屏障通透性增高,还是两种受体都参与其中,尚不十分清楚。因此我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型,观察TNFα对肠上皮细胞屏障通透性、细菌穿过肠上皮屏障的数量和肠上皮细胞间紧密连接的影响,并在此基础上探讨TNFα影响紧密连接的机制和可能的信号通路,以明确病理条件下,TNFα增加细菌定位转移和肠上皮细胞屏障通透性的作用机制。材料和方法1、体外肠上皮细胞屏障模型的建立应用Caco-2细胞株建立体外肠上皮细胞屏障模型,并应用跨上皮细胞电阻（TEER）和荧光黄透过率两种指标检测肠上皮细胞屏障的完整性。2、TNFα对肠上皮细胞屏障通透性的影响不同浓度TNFα（0、10、50、100 ng/ml）作用24h或TNFα（100 ng/ml）作用不同时间（2、4、8、24h）,观察TNFα作用前后所引起的Caco-2细胞屏障跨上皮细胞电阻（TEER）的改变,以及TNFα对荧光黄透过率的影响。3、TNFα对大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的影响不同浓度TNFα（0、10、50、100 ng/ml）作用24h或TNFα（100 ng/ml）作用不同时间（2、4、8、24h）,观察TNFα对耐利福平大肠杆菌E coli K1.E44穿越肠上皮细胞屏障数量的影响。4、TNFα对肠上皮细胞间紧密连接结构的影响应用透射电镜观察TNFα（0、50、100 ng/ml）作用于Caco-2细胞24h后所引起的细胞间紧密连接超微结构形态学的改变。5、TNFα对肠上皮细胞紧密连接蛋白ZO-1表达和定位的影响应用免疫荧光、蛋白印迹杂交（Western blot）和实时定量PCR技术检测不同浓度的TNFα（0、10、50、100 ng/ml）作用于Caco-2细胞24h后所引起的肠上皮细胞紧密连接蛋白ZO-1定位和表达的变化情况。6、抗TNFRⅠ、RⅡ抗体阻断TNFα对肠上皮细胞屏障的影响加入TNFα100ng/ml作用前30min先加入抗TNFRⅠ、RⅡ单克隆抗体（2μg/ml）封闭细胞表面的TNF受体,在TNFα作用24h后,分别检测TEER、荧光黄透过率、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的活菌数量;并应用免疫荧光和Western blot技术检测抗TNFRⅠ、RⅡ抗体封闭细胞表面的TNF受体后,TNFα对肠上皮屏障的作用。结果1、TNFα增加肠上皮细胞屏障的通透性与对照组相比,50ng/ml TNFα可明显降低肠上皮细胞屏障的TEER（p＜0.001）,增加荧光黄的透过率（p＜0.0005）,100ng/mlTNFα这种作用更加明显（p＜0.0005）。TNFα100ng/ml作用4h后,TEER值即开始下降（p＜0.05）,荧光黄透过率开始升高（p＜0.001）,24h这种作用达到最强（p＜0.005）。2、TNFα增加大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量TNFα作用于Caco-2细胞单层24h,使细菌定位转移数量明显,且转移的活菌数量与TNFα剂量相关。TNFα（100ng/ml）时为6.83±0.16log₁₀CFU/ml,与没有TNFα刺激时定位转移活细菌数相比增加了近千倍（2.05±0.18log10CFU/ml,p＜0.001）。3、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量增加与肠上皮细胞屏障的通透性增加相关TNFα（100ng/ml）作用于Caco-2细胞的24h观察时间内,作用时间越长,对TEER、细菌定位转移和荧光黄透过率的影响就越大,表现出时间依赖性。分析两两因素之间的相互关系时,荧光黄透过率和穿过Caco-2细胞单层的活细菌数量之间（r=0.991）、TEER和荧光黄透过率之间（r=-0.998）以及TEER和穿过Caco-2细胞单层的活细菌数量之间（r=-0.996）呈现直线相关性（p均＜0.001）,提示这三个因素在时间上的变化是相一致的。4、TNFα破坏肠上皮细胞间的紧密连接在扫描电镜下观察,正常CaCo-2细胞,紧密连接结构完整,呈高电子密度的致密的带状结构。加入TNFα100ng/ml作用24h后,紧密连接结构电子密度降低,紧密连接变短,细胞缝隙增宽。5、TNFα引起肠上皮细胞紧密连接蛋白ZO-1的定位异常正常Caco-2细胞,ZO-1蛋白沿细胞膜分布;加入TNFα10ng/ml,ZO-1蛋白呈锯齿状分布,并且荧光信号减弱;加入TNFα100ng/ml,荧光信号进一步减弱,并且在胞浆内可见阳性染色,相邻细胞间缝隙增宽。6、TNFα引起ZO-1蛋白表达减少与对照组比较,加入TNFα100ng/ml作用24h后,ZO-1蛋白的相对含量明显减少（0.14±0.02 vs 0.37±0.05,p＜0.05）。ZO-1蛋白的表达还受到TNFα作用时间的影响,有时间依赖性。7、TNFα不影响ZO-1 mRNA的表达与对照组比较,不同浓度的TNFα或TNFα（100ng/ml）作用不同时间,没有引起ZO-1 mRNA的明显改变（p=0.85）。8、抗TNFRⅠ、RⅡ抗体阻断TNFα增加肠上皮细胞屏障通透性的作用与TNFα（100ng/ml）相比较,抗TNFRⅠ、RⅡ抗体（2μg/ml）预先封闭细胞表面的受体后,Caco-2细胞单层TEER%上升（76.9±11.88%、72.79±5.21%vs40.3±7.2%,p＜0.001）。荧光黄透过率明显减少（12.6±0.81%和14.2±0.83%vs31.5±3.93%,p＜0.0001）。9、抗TNFRⅠ、TNFRⅡ抗体减轻TNFα促进大肠杆菌肠E coliK1.E44穿越肠上皮细胞屏障的作用与TNFα（100ng/ml）相比较,抗TNFRⅠ、RⅡ抗体（2μg/ml）预先封闭细胞表面的受体后,细菌转移数量明显下降(3.51±0.49log₁₀CFU/ml、3.82±0.46log₁₀CFU/ml vs 6.83±0.16log₁₀CFU/ml,p＜0.0001)。10、抗TNFRⅠ、TNFRⅡ抗体阻断TNFα下调ZO-1蛋白表达的作用与TNFα（100ng/ml）相比较,封闭细胞表面TNFRⅠ、TNFRⅡ受体后ZO-1蛋白的表达增加（0.28±0.03、0.32±0.06 vs 0.21±0.015,p≤0.05）。结论1、TNFα增加肠上皮细胞屏障的通透性。2、TNFα增加大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量。3、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量增加与肠上皮屏障通透性相关,提示大肠杆菌的定位转移与肠上皮细胞的通透性增加有关。4、TNFα破坏肠上皮细胞间的紧密连接,这是其增加肠上皮细胞屏障细胞旁通透性的作用位点。5、TNFα引起紧密连接蛋白ZO-1表达减少,并发生定位变化,从细胞膜向胞浆转移。6、TNFα不影响ZO-1 mRNA的表达,提示TNFα对ZO-1的调节发生在蛋白水平。7、抗TNFRⅠ、TNFRⅡ单克隆抗体能够部分阻断TNFα对肠上皮屏障的作用,提示TNFα是通过TNFRⅠ、TNFRⅡ发挥对肠上皮细胞紧密连接的作用的。更多还原

【Abstract】 ObjectiveIn addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Breakdown of the barrier is implicated in bacterial translocation,leading to sepsis,and in the pathogenesis of several illnesses such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.Studies in rodents show that tumor necrosis factorα(TNFα) can lead to increased ileal permeability,and anti-TNFαantibody can prevent the intestinal permeability disorders.The mechanisms of TNFα-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via the interepithelial tight junction(TJ) proteins, and subsequent stimulation of highly immunoreactive submucosal cells are likely to play a significant role.Therefore,we detect the effect of TNFαon intestinal permeability,bacteria translocation and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells,and study the mechanisms involved in tight junction induced by TNFα.Materials and methods1、Cell culturesCaco-2 cells were grown in a culture medium composed of RPMI 1640 with 50IU/ml penicillin,50μg/ml streptomycin and 15%FBS.In order to establish in vitro intestinal epithelial barrier model,Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by epithelial resistance measurements. 2、Determination of epithelial monolayer resistance,paracellular permeability and the clone forming units per milliliter of E coli K1.E44The electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using the millicell-ERS as previously reported.For determination of the effect of TNFαon Caco-2 monolayer paracellular permeability,the filter-grown Caco-2 cells by 3 wk postplating were incubated with TNFα(0,10,50 or 100ng/ml) for 24 hours or with TNFα100ng/ml for indicated times(2,4,8,24h).The transepithelial resistance,mucosal-to-serosal flux rate of the established paracellular marker lucifer yellow and bacterial translocation was assessed with or without pretreatment with anti-TNFRⅠmonoclonal antibody or anti-TNFRⅡmonoclonal antibody.3、Transmission electron microscopyCells were treated with TNFα(0,50 or 100ng/ml) for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold’s lead citrate. Ultrastructure of junctional complexes was observated by transmission electron microscopy.4、Localization and expression of ZO-1 proteinCells were treated with TNFα(0,10,50 or 100ng/ml) for 24 hours and the localization and expression of ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR-Green-based real-time PCR was used to measure the expression of ZO-1 mRNA.Results1、TNFαincrease intestinal epithelial paracellular permeability and bacterial translocationTNFαtreatment of filter-grown Caco-2 monolayers(0-100ng/ml) produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period.The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers lucifer yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to lucifer yellow.The enhanced bacterial translocation was observed when confluent Caco-2 cell monolayers were exposed to TNFα.Blocking antibodies showed that both anti-TNFRⅠand TNFRⅡmonoclonal antibodies decreased intertinal epithelial paracellular permeability and bacterial translocation.2、TNFαdisrupts the interepithelial tight junctionNormal tight junctions were visible at the apical portion of the lateral cell membrane in Caco-2 cells,showing as the high electron-dense material.In cells without TNFα,the TJ displayed an intact structure.When the cells were incubated with TNFα(50,100ng/ml) for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.3、TNFαcauses tight junction protein ZO-1 disturbanceIn the control Caco-2 monolayers,ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.TNFα(100ng/ml) caused a progressive disturbance in the continuity of ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contacts.Anti-TNFRⅠor anti-TNFRⅡmonoclonal antibody prevented the TNFα-induced location and expression disturbance of ZO-1.4、TNFαdecreases the expression of ZO-1 proteinThe effect of TNFαon ZO-1 protein expression was examined by Western blot analysis.TNFαproduced a progressive decrease in ZO-1 protein expression over the 24h treatment period.The maximal drop in ZO-1 protein level occurred at TNFα100ng/ml and at the 24h time point.Either anti-TNFRⅠor TNFRⅡmonoclonal antibodies prevented the TNFα-induced decrease in ZO-1 protein level.5、TNFαdoes not affect the expression of ZO-1 mRNATNFαdoes not cause a significant decrease in ZO-1 mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment.Conclusion1、TNFαdisrupted the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.2、TNFαcaused a disturbance in the continuity of ZO-1 localization at the cellular borders and produced a decrease in ZO-1 protein expression.3、TNFαdidn’t induced a significant decrease in ZO-1 mRNA production.4、Anti-TNFRⅠor TNFRⅡmonoclonal antibodie attenuated the effect of TNFαon the intestinal epithelial permeability,the tight junction constructure and tight junction protein expression.更多还原