【作者】 刘圣烜；

【导师】 黄志华；

【作者基本信息】 华中科技大学 ， 儿科学， 2010， 博士

【摘要】 第一部分急性肝内胆汁淤积性黄疸幼鼠肠黏膜屏障损伤机制的研究目的:为模拟人体淤胆型肝炎建立α-异硫氰酸萘酯(ANIT)诱导的急性肝内胆汁淤积性黄疸的幼鼠模型,研究急性肝内胆汁淤积性大鼠小肠粘膜屏障的变化及其机制。方法:雄性三周龄SD大鼠ANIT50mg/kg一次性灌胃,建立急性肝内胆汁淤积动物模型,于灌胃后24、48、72小时观察各组大鼠血清总胆红素(TB)和丙氨酸转氨酶(ALT)的浓度,并采用免疫组化和Western blots免疫印迹法检测末端回肠黏膜紧密连接蛋白ZO-1(Zonula occludens-1),闭锁蛋白(Occludin),以及两种细胞因子核因子-κB (NF-κB)和肿瘤坏死因子-α(TNF-α)的分布和表达,并利用图像分析系统对Western blots图像结果进行定量分析。结果:1.灌服ANIT后,模型组大鼠血清ALT、TB逐渐升高,24h增加明显,48h达到高峰,72h开始下降。各时间点与对照组相比有明显差异(P<0.01)。2. ZO-1和Occludin蛋白主要沿大鼠小肠黏膜上皮细胞膜的顶端呈线状分布,模型组大鼠24h时ZO-1和Occludin的阳性染色较对照组减少,48h减少最为明显,72h阳性染色有所恢复。Western blots结果与免疫组织化学结果相一致。各时间点与对照组相比有明显差异(P<0.01)。3.模型组大鼠NF-κB和TNF-α蛋白表达与ZO-1和Occludin蛋白的表达呈负相关,于24h开始升高,48h达到最高点,72h开始有所下降。Western blots结果与免疫组织化学结果相一致。各时间点与对照组相比有明显差异(P<0.01)。结论:ANIT诱导的急性肝内胆汁淤积发生时,NF-κB受刺激激活,导致炎症介质TNF-α释放增加,TNF-α可引起其他炎症介质释放增加并进一步再活化NF-κB,触发炎症介质的“瀑布样级联反应”从而导致炎症反应。由NF-κB和TNF-α介导的炎症反应引起小肠黏膜上皮紧密连接蛋白分布异常及数量改变,影响肠黏膜上皮屏障的完整性,破坏小肠黏膜屏障。第二部分益生菌对急性肝内胆汁淤积性黄疸幼鼠肠粘膜屏障影响目的:研究双歧三联活菌(培菲康)对急性肝内胆汁淤积性大鼠小肠上皮细胞紧密连接蛋白ZO-1和Occludin表达的影响以及对细胞因子NF-κB和TNF-α表达的影响,从而探讨益生菌制剂对急性肝内胆汁淤积性黄疸幼鼠肠粘膜屏障的保护机制,为益生菌制剂在临床中的应用提供理论依据。方法:雄性三周龄SD大鼠随机分为对照组、模型组和培菲康组,模型组和培菲康组均给予ANIT50mg/kg一次性灌胃,建立急性肝内胆汁淤积动物模型,培菲康组于造模前4天开始给予培菲康4.2×108个活菌数/kg/d灌胃大鼠。分别于造模后24、48、72小时三个时间点处死大鼠,取腹主动脉血检测血清TB和ALT水平,并取末端回肠黏膜组织,采用免疫组化和Western blots免疫印迹法检测紧密连接蛋白ZO-1, Occludin,以及细胞因子NF-κB和TNF-α的分布和表达,并利用图像分析系统对Western blots图像结果进行定量分析。结果:1.各时间点培菲康组大鼠血清ALT、TB较模型组明显降低(P<0.05)。2.模型组大鼠24h时ZO-1和Occludin的阳性染色较对照组减少,48h减少最为明显,72h阳性染色有所恢复,而培菲康组大鼠各时间点ZO-1和Occludin的阳性染色和模型组相比均明显增多。Western blots结果与免疫组织化学结果相一致,模型组24h已经开始下降,48h达到最低,72h开始恢复,各时间点与对照组相比有明显差异(P<0.01);而培菲康组各时间点蛋白表达与模型组相应时间点数据相比有明显恢复(P<0.05)。3.模型组大鼠NF-κB和TNF-α蛋白表达较对照组于24h开始升高,48h达到最高点,72h开始有所下降。Western blots结果与免疫组织化学结果相一致。各时间点与对照组相比有明显差异(P<0.01)。培菲康组大鼠NF-κB和TNF-α的蛋白表达与模型组大鼠在各时间点相比均明显降低(P<0.05)。结论:双歧三联活菌能够增加急性肝内胆汁淤积性黄疸大鼠小肠黏膜上皮紧密连接蛋白ZO-1和Occludin的表达,同时能降低NF-κB和TNF-α两种细胞因子在肠道内的表达,从而降低肠道内炎症反应发生的几率,有助于恢复肠黏膜上皮屏障的完整性。更多还原

【Abstract】 Part IInvestigation of the mechanism of the damage of intestinal mucosal barrier in rats with acute intrahepatic cholestasisObjective: To investigate the changes of intestinal mucosal barrier in the rat models with acute intrahepatic cholestasis induced by alpha-haphtylisocyocyanate(ANIT), and explore its mechanisms.Methods: A single dose (50 mg/kg) ofα- naphthylisothiocyanate (ANIT) was administered by gavage to each experimental rat to induce intrahepatic cholestasis. At 24h、48h and 72h after gavage, the levels of the serum total bilirubin (TB) and alanine aminotransferase(ALT) were detected and immunohistochemistry and western blot techniques were used to examine the distribution and expression of tight junction proteins—Zonula occludens-1 (ZO-1)、Occludin and two cell factors—nuclear transcription factor-kappa B (NF-κB)、tumor necrosis factor (TNF-α). And the results of Western blot were quantitative analysised by image analytical system.Results:1. After the administration of ANIT, the levels of serum ALT and TB in the model group increased gradually, reached the pinnache at 48h.The levels of serum ALT and TB in the model group at each time point were obviously higher than those in control group (P<0.01).2. ZO-1 and Occludin were localized along the apical region of the lateral plasma membrane representing the region of tight junctions in surface and crypt epithelial cells. The positive stainings of ZO-1 and Occludin of model group at 24h after gavage, were decreased than those of control group, the decrease was most obviosly at 48h, and partly recovered at 72h. Western blot demonstrated consistent and significant reduction with immunohistochemistry. The protein expressions in model group were significantly lower than those in control group(P<0.01).3. The expressions of NF-κB and TNF-αin model group were inverse correlation with the expressions of ZO-1 and Occludin. The expressions of NF-κB and TNF-αin model group were markedly higher than those in control group.Conclusion: When Acute intrahepatic cholestasis induced by ANIT happened, NF-κB was activated and made the levels of TNF-αincreased. TNF-αcould release other mediators of inflammation and reactivated NF-κB. These could induce inflammatory reaction and the abnormal distribution of tight junction proteins and the alteration of their quantity, which affects the intestinal barrier integrity, resulting in impaired barrier function.Part IIEffects of probiotics on the intestinal mucosal barrier in rats with experimental acute intrahepatic cholestasisObjective: To study the effects of live combination bifidobacterium, lactobacillus and enterococcus(BIFICO) on the expressions of intestinal mucosal epithelium tight junction protein—ZO-1、Occludin and cytokine—NF-κB、TNF-αin rats with cholestasis and further discuss the protection of the probiotics to intestinal mucosal barrier, so as to provide the academic basis for its clinical uses.Methods: Male SD rats of 3 weeks old were randomly divided into control group, model group and BIFICO group. Before making the animal model, BIFICO(4.2×108 viable bacterium counts/kg/d) were adminsterated to the rats in BIFICO group for 4 days. Model and control groups were fed by normal saline. All groups did not stop being administrated treating agent daily until executed. At the 5th day after administration, after fasting for 12h, model group and BIFICO group were intragastrically administrated ANIT(50mg/kg) for modeling. At 24h、48h and 72h after modeling, every 10 rats in each group were executed for taking abdominal aortal blood and terminal ileum tissue. The levels of serum TB and ALT were detected and immunohistochemistry and western blot techniques were used to examine the distribution and expression of tight junction proteins—ZO-1、Occludin and cell factors—NF-κB、TNF-α. And the results of Western blot were quantitative analysised by image analytical system.Results:1. The levels of serum ALT and TB in the BIFICO group at each time point were obviously lower than those in model group(P<0.05).2. The positive stainings of ZO-1 and Occludin of model group at 24h after modeling, were decreased than those of control group, the decrease was most obviosly at 48h, and partly recovered at 72h. Western blot demonstrated consistent and significant reduction with immunohistochemistry. The protein expressions in BIFICO group have significant recovery compared to those in model group at each time point(P<0.05).3. The protein expressions of NF-κB and TNF-αin BIFICO group were notably lower than those in model group at each time point(P<0.05). Conclusion: The results suggested that probiotics supplement can improve the expressions of ZO-1 and Occludin and at the same time decrease the expressions of NF-κB and TNF-αin rats with acute intrahepatic cholestasis, thereby it can cut down the probability of intestinal inflammatory reaction and conduce to improve the intestinal barrier function.更多还原