【作者】 邓郁青；

【导师】 魏林；

【作者基本信息】 河北医科大学 ， 免疫学， 2014， 博士

【摘要】 目的：应激是指机体在受到内外环境因素及社会、心理因素刺激时所出现以交感神经系统兴奋和下丘脑－垂体－肾上腺轴激活，皮质激素分泌增多为主的一系列神经内分泌反应，是一种全身性的非特异适应反应。越来越多的证据显示应激可以增加包括传染性病原体导致的炎症性疾病在内的多种疾病的发病率。免疫系统作为身心关系的桥梁，其在应激时所受到的影响越来越受到人们的重视，应激对机体免疫功能的影响已成为目前研究的热点之一。应激在功能性胃肠道疾病发病及疾病进展中的作用机制是目前基础和临床医学研究中的重要课题之一。应激与多种功能性胃肠道疾病的发生发展密切相关。胃肠道不仅是机体消化吸收营养物质的场所,也是应激时的重要靶器官，同时还是体内重要的免疫组织器官，发挥着重要的免疫防护作用。由完整的上皮细胞、上皮细胞刷状缘的脂质双分子层以及细胞侧缘由黏着连接和紧密连接构成的细胞连接组成的肠道黏膜屏障具有机械屏障作用，可以有效阻挡病体侵入体内，是机体固有免疫系统抵御外来异物入侵的第一道防线之一，肠相关淋巴组织是发生局部特异性免疫应答的主要场所，肠黏膜固有层浆细胞产生的分泌型IgA使得肠道免疫成为全身免疫的一部分，存在于胃肠道的多种免疫细胞一方面通过其细胞膜上分布的多种受体接受神经内分泌系统调节，另一方面可以通过分泌多种细胞因子作用于其它免疫细胞及神经内分泌系统。鲁比前列酮（Lubiprostone），是一种局部作用的Ⅱ型氯离子通道激活剂，最初用于治疗成人慢性特发性便秘，后来又被批准用于治疗成人肠易激综合征。心理应激导致的免疫系统改变在不同的时间点，其作用模式有何不同？在慢性心理应激导致胃肠道疾病发生的过程中，肠道黏膜机械屏障是否受到损伤？肠道免疫屏障是否会随之受到影响？鲁比前列酮可否用于应对心理应激导致的肠道屏障异常？为深入研究心理应激对机体免疫系统的影响，本研究以SD大鼠为研究对象，建立心理应激动物模型，在不同时间点分别检测心理应激对机体全身免疫和肠道局部免疫所造成的影响，在此基础上，进一步探讨心理应激导致肠道局部黏膜改变的靶分子，研究鲁比前列酮对其发挥作用的可能性和可能的作用机制。方法：1影响肠道功能的心理应激动物模型的建立及心理应激对免疫系统的动态影响我们首先建立了可以影响肠道功能的心理应激动物模型，应用此动物模型动态观察在心理应激发生的不同阶段机体免疫系统可能发生的改变，明确心理应激在不同时间点对免疫系统的影响模式。以SD大鼠为研究对象，建立心理应激作用动物模型，观察心理应激对大鼠一般状况的影响，通过对大鼠排便情况的观察和对内脏敏感性的检测明确WAS是可以影响大鼠肠道功能的心理应激动物模型。在不同的时间点收集大鼠血清，应用ELISA法检测血清中循环细胞因子IL-1β、IL-2、IFN-γ、IL-6和TNF-α的表达水平，明确急性和慢性心理应激对机体全身免疫可能造成的影响。分离大鼠结肠，应用高通量qPCR来初步筛选慢性心理应激可能会影响的免疫分子、黏附分子、神经递质等，为下一步研究指明方向。2心理应激对大鼠肠道局部免疫功能的动态影响在第一部分实验的基础上，继续应用WAS动物模型，明确心理应激在不同的时间点对肠道局部免疫的影响模式。应用实时定量PCR和Western Blot方法检测不同的时间点局部结肠组织中IL-1β、IL-2、IFN-γ、IL-6、IL-10和TNF-α等细胞因子的表达水平。应用ELISA检测肠液中SIgA的水平。3慢性心理应激及鲁比前列酮对大鼠肠上皮紧密连接蛋白的改变及其作用机制在明确心理应激对肠道免疫系统的动态影响后，进一步研究慢性心理应激及鲁比前列酮对大鼠肠上皮紧密连接蛋白的改变及其作用机制。筛选最佳的药物作用剂量，使用该剂量在假应激或慢性心理应激之前给予大鼠口服鲁比前列酮。连续10天WAS结束后，分离大鼠肠道上皮细胞和肠液。应用实时定量PCR检测鲁比前列酮对慢性心理应激大鼠肠上皮紧密连接分子转录水平的影响。应用Western Blot检测鲁比前列酮对慢性心理应激大鼠肠上皮紧密连接蛋白表达水平的影响。应用免疫荧光法检测鲁比前列酮对慢性心理应激大鼠肠上皮紧密连接蛋白形态学的影响。应用ELISA检测鲁比前列酮作用后慢性心理应激大鼠肠液中SIgA的水平变化。结果：1影响肠道功能的心理应激动物模型的建立及心理应激对免疫系统的动态影响慢性心理应激可以显著抑制大鼠的体重增长，应激原去除后，其抑制作用随着时间的推移可以消失。心理应激可以影响自主神经系统对远端结肠动力的调控，刺激大鼠排便，能够显著提高实验动物的内脏痛觉敏感。与对照组相比，急、慢性心理应激可以在不同时间段引起不同细胞因子在血清中的表达水平的改变，其中，1次避水应激后1小时时大鼠血清中IL-1β、IL-6、IFN-γ显著上升（P<0.05），1次避水应激后24小时时，IL-1β、IL-6显著上升（P<0.05），而慢性应激对IL-1β、IL-2、IL-6、IFN-γ、TNF-α表达水平的影响无显著差异（P>0.05）。高通量筛选显示慢性心理应激可以导致结肠中多种分子转录水平改变，上调的主要有Adenosine A1receptor、BDNF、CCL12、CD200、Claudin2、CDK2、HDAC2、HDAC4、IL-6、Integrin-alpha M、P2RX3等，出现下调的有Aurora kinase A、BAZ1b、 Claudin1、Claudin6、CB2、CSF1、DMNT1、PRMT1。2心理应激对大鼠肠道局部免疫功能的动态影响实时定量PCR检测显示急性心理应激（1次避水应激后1小时、24小时）及慢性心理应激可以导致不同时间点不同细胞因子的mRNA水平发生改变。其中，急性、慢性心理应激均可导致大鼠结肠IL-1β、IL-6表达水平上升（P<0.05），1次避水应激后1小时、24小时时大鼠结肠TNF-α表达水平上升（P<0.05），1次避水应激后24小时时大鼠结肠IL-2、IL-10表达水平上升（P<0.05），慢性心理应激对IL-2、IL-10、TNF-α和IFN-γ表达水平无显著影响（P>0.05）。Western Blot检测显示急性心理应激及慢性心理应激均可导致大鼠结肠IL-1β、IL-6表达水平上升（P<0.05），1次避水应激后24小时及慢性心理应激均可导致大鼠结肠IL-2表达水平上升（P<0.05），急性心理应激（1次避水应激后1小时、24小时）后，大鼠结肠TNF-α表达水平上升（P<0.05），急、慢性心理应激对IL-10、IFN-γ的均无显著影响（P>0.05）。该结果与实时定量PCR检测结果基本平行。ELISA检测显示急性心理应激（1次避水应激后1小时、24小时）后，大鼠结肠肠液中SIgA水平上升（P<0.05），慢性心理应激对肠液中SIgA的影响无统计学意义（P>0.05）。3慢性心理应激及鲁比前列酮对大鼠肠上皮紧密连接蛋白的改变及其作用机制首先通过对内脏敏感性的检测实验确定鲁比前列酮的最佳使用剂量为20μg/kg/day，每天两次给药。随后应用实时定量PCR和Western Blot方法检测发现慢性心理应激可以在转录水平和蛋白水平显著下调结肠上皮细胞紧密连接蛋白ZO-1、Claudin-1、Occludin的表达，镜下观察可以发现对照组紧密连接蛋白在上皮的顶端膜侧表达，在肠上皮黏膜层分布均匀，分布广泛，荧光信号明显，表达量丰富，而慢性应激组的荧光信号减弱，呈不连续分布，鲁比前列酮可以部分逆转慢性心理应激导致的紧密连接蛋白Claudin-1和Occludin表达下降，但对ZO-1蛋白无显著作用。慢性心理应激可以导致结肠上皮细胞Claudin-2的表达出现上调，这种上调和ZO-1的下调一样无法被鲁比前列酮所逆转结论：1慢性心理应激可以增加大鼠肠道痛觉敏感性。2急性心理应激上调血清中IL-1β、IL-6的表达，急性心理应激和慢性心理应激均可以显著上调肠道局部IL-1β、IL-6的表达。3慢性心理应激可以显著下调结肠上皮细胞紧密连接蛋白ZO-1、Claudin-1、Occludin，上调Claudin-2的表达，破坏肠道黏膜屏障。鲁比前列酮可以部分逆转慢性心理应激导致Claudin-1和Occludin表达下降。4急性心理应激可以导致大鼠结肠液中SIgA水平上升，慢性心理应激和鲁比前列酮对肠液中SIgA水平无显著影响。更多还原

【Abstract】 Objective: Stress is a general body response to initially threateningexternal or internal demands, involving the mobilization of physiological andpsychological resources to deal with them. The major mediators of stresseffects are the sympathetic nervous system and corticotrophin releasinghormone, adrenocorticotropin, and cortisol, which arise following activationof the hypothalamic-pituitary-adrenal axis. It is a proven fact that stressincreases the susceptibility to inflammatory disorders, including those ofinfectious etiology. The magnitude and duration of stress-induced elevationsin catecholamine and glucocorticoid hormones can have significant effects onimmune cell distribution and function. More and more people focus on thei n f l u e n c e o n t h e i m m u n e s y s t e m b y t h e s t r e s s.The association of acute and chronic stress related with the susceptibilityto the functional gastrointestinal disease was proved. Gastrointestine not onlyworks as the place for the absorption of nourishment, but also the importanttarget organ at the stress situation, as well as the important immune organ. Theintestinal mucosal barrier which builds up with the intestinal epithelial cells,tight junction, adhesion junction and gap junction is work as a mechanicalbarrier. It acts as the first line to block the invasion from the pathogen. TheSecretory immunoglobulin A (S-IgA) is a member of the adaptive secretoryimmune system, which made the intestinal immune system works togetherwith the other immune system. Receptors of glucocorticoids andcatecholamines are found on almost all the immune cells, the increased plasmaand tissue concentrations of these hormones during stressful conditions alterthe function of these cells, at the same time; immune cells can affect then e u r o e n d o c r i n e s y s t e m b y r e l e a s i n g t h e c y t o k i n e.Lubiprostone is a novel drug for adult chronic idiopathic constipation Which investigated by Sucampo and Takeda pharmaceuticals company. Thisdrug can selective active the ClC-2channel which exist at the gastrointestinaltract epithelium tissue, increase the intestinal motive. It was found thatLubiprostone can prove the visceral hyperalgesia.Is the stress-induced change at the immune system depended on thetimeline? At the period of the gastrointestinal disease which induced by thechronic psychological stress, the mechanical barrier is damaged or not? Howabout the immune barriers? Can Lubiprostone reverse the dysfunction whichinduced by the chronic psychological stress?To answer these questions, we use SD rats to set up the animal model.The immune targets were examined at the different time point. Base of theresults, we try to find the target molecular during this change, and the possiblemechanism of the Lubiprostone.Methods：1Set up the stress-induced animal model which can affect thegastrointestinal function. The model would be used to study the changeswhich induced by stress. A time course should be draw from these results.Set up the animal models, observe the general condition of the rat whichaffect by stress. The observation about the fecal pellet output and the VMR toCRD can determine the model’s usage.The serum of rats was collected at different time point. The circulatingcytokine IL-1β、IL-2、INF-γ、IL-6、TNF-β level were detected by ELISA. Theresults used to setup a timeline about the stress-induced change at theintestinal immune system.SuperArray was used to detect the level of many kinds ofneurotransmitter, kinase and immune molecular which can be affected bychronic stress2the dynamic effect on the rats’ immune function by psychologicalstressReal-time PCR and Western Blot were used to examine the tissuecytokine IL-1β、IL-2、INF-γ、IL-6、TNF-β which gathered at different time. The SIgA of the intestinal juice was examined by ELISA3the effect of the enteric epithelium enteric epithelium protein whichinduced by chronic stressThe dose was determined according the paper from Takera and results.The rat were feed Lubiprostone everyday during the WAS.The enteric epithelium tight junction molecular of the rats was examinedby Real-time PCR.The enteric epithelium tight junction proteins of the rats were examinedby Western Blot and Immumofluorescence.The SIgA of the chronic–stress rat which treated by Lubiprostone wasexamined by ELISAResults:1Set up the stress-induced animal model which can affect thegastrointestinal function. The model would be used to study the changeswhich induced by stress. A time course should be draw from these results.Chronic stress inhibits the rat gain weight; the inhibitory action woulddisappear in several weeks.Compare with the control group, the serum level of IL-1β、IL-6、INF-γ at1h after1WAS were significantly increased, as well as the serum level ofIL-1β、IL-6at24h after1WAS were significantly increased, the serum level ofIL-1β、IL-2、IL-6、INF-γ、TNF-α at24h or72h after10WAS were nosignificantly increased. Compare with the control group.DNA assay shows some colonic molecular were upgrade, such asAdenosine A1receptor、BDNF、CCL12、CD200、Claudin2、CDK2、HDAC2、HDAC4、IL-6、Integrin-alpha M、P2RX3. Aurora kinase A、BAZ1b、Claudin1、Claudin6、CB2、CSF1、DMNT1、PRMT1and some others weresignificantly decreased after the chronic stress.2the dynamic effect on the rats’ immune function by psychologicalstressReal-time PCR shows the colonic level of IL-1β、IL-6were significantlyincreased after the acute or chronic stress, the colonic level of IL-2and IL-10 were significantly increased at24h after1WAS, the colonic level of TNF-αwere significantly increased at1h after1WAS and24h after1WAS, thecolonic levels of INF-γ were no significantly increased which compared withthe control group.Western Blot shows the colonic level of IL-1β、IL-6were significantlyincreased after the acute or chronic stress, the colonic level of IL-2weresignificantly increased at24h after1WAS and24h after1WAS, the colonlevel of TNF-α were significantly increased at1h after1WAS and24h after1WAS, the colonic level of INF-γ and IL-10were no significantly increasedafter the acute or chronic stress. Most of the results match the Real-time PCR.ELISA shows the level of SIgA was significantly increased at1h and24hafter1WAS, the level of SIgA was no significantly increased after the chronicstress treatment.3The effect of the enteric epithelium enteric epithelium protein whichinduced by chronic stressWestern Blot shows the level of the enteric epithelium tight junctionprotein ZO-1、Claudin-1、Occludin were significantly decreased after thechronic stress, the level of Claudin-2were significantly increased after thechronic stress. Lubiprostone may partly reverse the change of Claudin-1orOccludin by chronic stress treatment.ELISA shows the level of SIgA was no significantly change after thechronic stress and the Lubiprostone treatment.Conclusions:1Chronic psychological stress induces the rat’s visceral hyperalgesia.2Acute stress induces the serum level of IL-1β and IL-6weresignificantly increased, both acute and chronic stress induces the colon levelof IL-1βand IL-6were significantly increased.3The level of the enteric epithelium tight junction protein ZO-1,Claudin-1, Occludin were significantly decreased after the chronic stress, thelevel of Claudin-2were significantly increased after the chronic stress, whichlead to the damage of the mucosal barrier. Lubiprostone may partly reverse the change of Claudin-1and Occludin which caused by chronic stresstreatment.4Acute stress induces the level of SIgA was significantly increased; thelevel of SIgA was no significantly change after the chronic stress and theLubiprostone treatment.更多还原