【作者】 崔巍；

【导师】 刘沛；

【作者基本信息】 中国医科大学 ， 内科学传染病， 2007， 博士

【摘要】 肿瘤坏死因alpha增加肠上皮细胞屏障通透性的机制研究目的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。目前研究发现肠上皮细胞屏障通透性增高参与多种疾病的发生(如炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等)，并且在这些情况下，血清肿瘤坏死因子alpha(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。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凋亡非依赖性的。另外TNFα引起肠上皮细胞屏障通透性增高的具体作用环节也不十分清楚，可能与蛋白激酶C(PKC)、核因子kB(NF-kappaB)、肌球蛋白轻链激酶(MLCK)、丝裂原活化的蛋白激酶(MAPK)等信号途径有关。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和肠上皮细胞间紧密连接的影响，并在此基础上探讨了TNFα对调控紧密连接装配的蛋白酶活化受体-3／非典型蛋白激酶C／蛋白酶活化受体-6(Par-3／aPKC／Par-6)复合体的影响和可能信号通路，以明确病理条件下，TNFα增加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形成情况。2、TNFα对肠上皮细胞屏障通透性的影响加入不同浓度的TNFα(10μ／L或100μg／L)作用24h或加入TNFα100pg／L作用不同时间(2、4、8、24h)，观察加入TNFα前后Caco-2细胞屏障跨上皮细胞电阻的改变及TNFα对Caco-2细胞荧光黄透过率的影响。3、TNFα对肠上皮细胞间紧密连接的影响透射电镜下观察加入TNFα100μtg／L作用24h后，Caco-2细胞间紧密连接形态学的改变。4、TNFα对肠上皮细胞紧密连接蛋白occludin定位和表达的影响制备Caco-2细胞NP-40可溶性和NP-40不溶性蛋白框架，应用蛋白印迹杂交(Western blot)技术，检测加入不同浓度的TNFα(10μg／L或100μg／L)作用24h后，不同蛋白框架内肠上皮细胞紧密连接蛋白occludin表达的变化情况。并应用免疫荧光和实时定量PCR技术检测occludin蛋白的定位和mRNA表达的变化情况。5、TNFα对肠上皮细胞Par-3表达和定位的影响加入TNFα100μg／L作用不同时间(0、4、8、24h)后，应用免疫荧光、Western blot和逆转录-聚合酶链反应(RT-PCR)技术检测TNFα对肠上皮细胞Par-3表达和定位的影响。6、TNFα对肠上皮细胞非典型蛋白激酶C亚型PKλ丸和PKCζ活性的影响应用抗PKCζ／λ(Thr410／403)磷酸化抗体检测加入TNFα100μg／L作用不同时间(0、4、8、24h)后，PKCλ／ζ(活性的改变。7、应用免疫共沉淀的方法检测Caco-2细胞内与Pat-3结合的信号分子应用抗Par-3抗体沉淀Caco-2细胞裂解产物，然后应用抗T淋巴瘤侵袭转移因子(Tiaml)抗体对沉淀产物进行蛋白印迹杂交，明确Par-3是否与Tiaml结合。8、TNFα对肠上皮细胞T淋巴瘤侵袭转移因子表达和定位的影响加入TNFα100μg／L作用不同时间(0、4、8、24h)后，应用免疫荧光、免疫电镜、Western blot和RT-PCR技术检测TNFα对肠上皮细胞Tiaml表达和定位的影响。9、TNFα对肠上皮细胞Racl活性的影响应用配体免疫沉淀法检测加入TNFα100μg／L作用不同时间(0、4、8、24h)后，Racl活性的改变。10、Tiaml在TNFα降低紧密连接蛋白occludin表达中的作用应用小核糖核酸干扰(siRNA)技术沉默Caco-2细胞内Tiaml基因，观察在Tiaml低表达的Caco-2细胞内，TNFα对紧密连接蛋白occludin表达的影响。结果1、TNFα增加肠上皮细胞屏障的通透性与对照组相比，10μg／L TNFα即可降低肠上皮细胞屏障的TEER(P＜0.0005)，增加荧光黄透过率(P＜0.0005)，100μg／L TNFα这种作用更加明显(P＜0.0005)。在作用时间上，TNFα100μg／L作用4h后，TEER值即开始下降(P＜0.0005)，荧光黄透过率开始升高(P=0.001)，24h这种作用达到最强(P＜0.0005)。2、TNFα破坏肠上皮细胞间的紧密连接正常CaCo-2细胞，紧密连接结构完整，呈致密的带状结构。加入TNFα100μg／L作用24h后，紧密连接断裂，细胞间隙增宽。3、TNFα引起肠上皮细胞紧密连接蛋白occludin的定位异常正常Caco-2细胞，occludin蛋白沿细胞膜分布；加入TNFα10μg／L作用24h后，occludin蛋白呈锯齿状分布，并且荧光信号减弱；加入TNFαt100μg／L作用24h后，锯齿状分布更加明显，荧光信号进一步减弱，并且在胞浆内可见阳性染色。4、TNFα引起occludin蛋白磷酸化减少与对照组比较，加入TNFα100μg／L作用24h后，NP-40不溶性蛋白框架内occludin蛋白(即磷酸化occludin蛋白)的相对含量明显减少(P=0.016)，而NP-40可溶性蛋白框架内occludin蛋白(即非磷酸化occludin蛋白)的相对含量没有变化(P=0.99)。5、TNFα不影响occludin mRNA的表达与对照组比较，不同浓度的TNFα或TNFα作用不同时间，均不能引起occludin mRNA的明显改变(P=0.85，P=0.99)。6、TNFα不影响Par-3的表达和定位正常Caco-2细胞，Par-3定位在细胞膜上，与紧密连接蛋白的定位一致，加入TNFα不能改变Par-3蛋白的定位。Western blot和RT-PCR结果提示加入TNFα后，Par-3蛋白和mRNA的相对含量与正常对照组比较没有差别(P=0.99，P=0.86)。7、TNFα下调PKCλ/ζ活性与正常对照组相比，TNFα100μg/L作用8h后，PKCλ/ζ活性开始下降(P=0.014)，24h达到最低(P=0.001)。8、Par-3与Tiam1免疫共沉淀应用抗Tiam1抗体对Par-3预沉淀产物进行蛋白印迹杂交后，发现在170kDa处出现特异性蛋白条带，证明在Caco-2细胞内，Par-3与Tiam1结合。9、TNFα增加Tiam1表达，促进Tiam1活化在正常Caco-2细胞，Tiam1分布在胞浆内，加入TNFα100μg/L作用24h后，Tiam1向细胞膜转移。Western blot和RT-PCR结果显示，与正常对照组比较，TNFα100μg/L作用24h后，Tiam1蛋白和mRNA的相对含量明显升高(P=0.029，P=0.011)。10、TNFα促进Rac1的活化与正常对照组比较，加入TNFα后，总Rac1的含量没有明显变化(P=0.99)。但活性Rac1(GTP-Rac1)的含量在TNFα100μg/L作用24h后明显升高(P=0.002)。11、Tiam1介导TNFα引起的紧密连接蛋白occludin磷酸化减少在Tiam1正常表达的Caco-2细胞，TNFα可以引起occludin蛋白磷酸化的减少，而在Tiam1低表达的Caco-2细胞，TNFα的这种作用消失，两者相比有明显的统计学差异(P=0.001)。1、TNFα增加肠上皮细胞屏障的通透性。2、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。3、TNFα引起紧密连接蛋白occludin的异常分布，使紧密连接蛋白occludin磷酸化减少，但不影响未磷酸化的occludin蛋白表达。4、TNFα不影响occludin mRNA的表达，提示TNFα对occludin的调节发生在蛋白水平，而不是转录水平。5、TNFα不影响Par-3的表达和定位，但能使PKCλ／ζ的活性降低，提示TNFα对于调控紧密连接装配的Par-3／aPKC／Par-6复合体的定位没有影响，但能抑制Par-3／aPKC／Par-6复合体的功能。6、Tiam1-Rac1是Par-3／aPKC／Par-6复合体的上游信号因子。7、TNFα能够增加Tiam1蛋白和mRNA水平的表达，促进Tiam1和Rac1活化。8、TNFα引起Caco-2细胞紧密连接蛋白occludin磷酸化减少需要Tiam1的参与。更多还原

【Abstract】 The mechanism of increased intestinal epithelial barrier permeability induced by tumor necrosis factor alphaObjectiveIn 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 factor alpha(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 and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells, and study the mechanisms involved in tight junction discharged assembly induced by TNFα.Materials and methods1、Cell culturesCaco-2 cells were grown in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mmol/1 glutamine, and 10% 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 and paracellular permeabilityThe electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohrn-meter (EVOM) 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 TNFα10μg/L or 100μg/L for 24 hours or with TNFα100μg/L for indicated times(2、4、8、24h), and the mucosal-to-serosal flux rates of the established paracellular marker luminal yellow was determined.3、Transmission electron microscopyCells were treated with TNFα100μg/L for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold’s lead citrate. Ultrastructure of junctional complexes were observated by transmission electron microscopy.4、Assessment of occludin protein localization and expressionCells were treated with TNFα(10μg/L or 100μg/L) for 24 hours and the localization and expression of occludin protein were detected by immunofluorescence and Western blot analysis. SYBR-Green-based real-time PCR was used to measure the expression of occluding mRNA.5、Assessment of Par-3 protein localization and expressionCells were treated with TNFα100μg/L for indicated hours(4、8、24h) and the localization and expression of Par-3 protein were detected by immunofluorescence and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Par-3 mRNA.6、PKCλand PKCξkinase analysisCells were treated with TNFα100μg/L for indicated hours(4、8、24h) and the endogenous PKCξ/λactivity was detected by immunoblotting with anti-phos-PKCξ/λ(Thr410/403) antibody.7、Immunoprecipitation In order to judge the endogenous complexes of Par-3 and Tiaml, Caco-2 cells lysates were immunoprecipitated with anti-Par-3 antibody and immunoprecipitates were immunoblotted with anti-Tiaml antibody.8、Assessment of Tiaml protein localization and expressionCells were treated with TNFα100μg/L for indicated hours (4、8、24h) and the localization and expression of Tiaml protein were detected by immunofluorescence, immunogold electron microscopy and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Tiaml mRNA.9、Racl activity assaysAfter treatment with TNFα100μg/L for indicated hours (4、8、24h), Caco-2 cells lysates were prepared and Racl activity was determined as described previously using a biotinylated Racl interactive binding metifpeptide of PAK1.10、RNA interference for Tiara1 geneSilence the Tiarnl gene in Caco-2 cells use small interfering RNA specific for Tiaml. Detect the effect of TNFαon action occludin protein expression in Tiaml knock down cells.Results1、TNFαincrease intestinal epithelial paracellularpermeabilityTNFαtreatment of filter-grown Caco-2 monolayers (0-100μg/L)produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period (P＜0.0005). The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers luminal yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to luminalyellow (P＜0.0005).2、TNFαdisrupts the interepithelial tight junctionThe presence of electron-dense material in the space between cells near the brush border reflects the TJ. In cells without TNFα, the TJ displayed an intact structure. When the cells were incubated with TNFα100μg/L for 24 hours, the TJ complex appeared reduced and contained less electron-dense material. 3、TNFαcauses tight junction protein occludin distributedIn the control Caco-2 monolayers, occludin proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders. TNFα(100μg/L) caused a progressive disturbance in the continuity of occludin localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact.4、TNFαdecreases the expression of action occludin proteinTNFαproduced a progressive decrease in higher molecular form, a phosphorylated form (85 kDa) of occludin protein expression (P=0.016), whereas the low molecular form, a nonphosphorylated form (65 kDa), was no significant change (P=0.99).5、TNFαdoes not affect the expression of occludin mRNATNFαtreatment does not cause a significant decrease in occludin mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment (P=0.85, P=0.99).6、TNFαdoes not affect the expression and localization of Par-3 proteinIn the control Caco-2 monolayers, Par-3 proteins were localized at the apical cellular borders, consistent with the location of occludin protein. TNFα(100μg/L) didn’t cause a significant change in the Par-3 protein localization. Western blot and RT-PCR analysis demonstrated that TNFαtreatment had on effect on the expression of Par-3 protein and mRNA (P=0.99, P=0.86).7、TNFαinhibits PKCλ/ζactivityTNFα100μg/L caused a progressive decrease in PKCλ/ζactivity over 8h period (P=0.014). The maxiam drop in PKCλ/ζactivity occurred at 24h (P=0.001).8、Par-3 immunoprecipitated with TiamlImmunoprecipitates with anti-Par-3 antibody showed specific protein belt by immunoblotted with anti-Tiaml antibody.9、TNFαincreases the expression of Tiaml proteinTiaml proteins were localized at the cytoplasm in the control Caco-2 cells. TNFα can cause a migration of Tiaml protein from cytoplasm to cellular borders. Furthrmore, Western blot and RT-PCR assays demonstrated that TNFαincresed the expression of Tiaml protein and mRNA at 24-h time point (P=0.029, P=0.011).10、TNFαstimulates Rac1 activityTNFαhad no significant effect on the expression of total Rac1 (P=0.99), whereas the active Rac1 (GTP-Rac1) decreased at 24-h time point after TNFαtreatment (P=0.002).11、TNFαdecreases action occludin protein expression through a Tiaml-dependent mannerTNFαdown regulated the phosphorylated form of occludin protein expression in Caco-2 cells. Moreover, absence of Tiaml rescued the impaired action occludin protein expression induced by TNFα(P=0.001).Conclusion1、TNFαdisrupts the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.2、TNFαcauses a disturbance in the continuity of occludin localization at the cellular borders and produces a decrease in action occludin protein expression.3、TNFαtreatment does not cause a significant decrease in occludin mRNA production.4、TNFαdoes not affect the expression and localization of Par-3 protein, but inhibits the activity of PKCλ/ξ.5、Tiaml-Pac1 is the upstream factor of Par3-aPKC-Par6 complex.6、TNFαcauses an increase in Tiaml protein and mRNA expression, and stimulates Rac1 constitutively activated.7、TNFαdownregulates action occludin protein expression via a Tiaml-dependent process.更多还原