【作者】 张茹；

【导师】 钱家鸣； 方秀才； 吕红；

【作者基本信息】 中国协和医科大学 ， 消化内科， 2008， 博士

【摘要】 研究背景:肠易激综合征(Irritable Bowel Syndrome,IBS)是一种常见的功能性肠病,其病因和发病机制并不清楚,目前公认IBS是多因素疾病。IBS病理生理学基础是肠道动力异常和内脏高敏感性。单胺类神经递质5-羟色胺(5-HT)、去甲肾上腺素(NE)是肠神经系统重要的神经递质,参与肠道感觉、动力、分泌的调控。本实验室前期在脑-肠交互作用理论基础上,建立了慢急性联合应激IBS大鼠模型,即动物在接受慢性不可预知轻度应激(CUMS)后接受一次急性束缚应激,已证实可从肠道动力、感觉和心理行为多方面模拟IBS表现,且具有稳定与重复性好的特点。本研究在前期对5-HT与NE在IBS动物模型研究的基础上,将应用慢急性联合应激动物模型完善对5-HT与NE系统的研究并进行新的探索研究,整个研究分为三部分。研究目的:1、通过测定各组大鼠感觉传导通路上脊髓背角、海马、额叶等脑—肠轴层面神经元c-fos水平,从分子水平评估慢急性联合应激动物模型。2、完善与深入探讨5-HT系统和NE系统在IBS的变化与可能的作用机制。3、利用蛋白质组学观察18S脑-肠交互作用动物模型结肠蛋白质全景,初步探讨IBS这一功能性肠病有无蛋白水平的变化。材料与方法:选用健康雄性成年Wistar大鼠,分为对照组、急性应激组(急性束缚1小时)、慢性应激组(CUMS,21天不可预知轻度应激)、慢急性联合应激组(联合应激组,CUMS的基础上给予急性束缚应激)。观察不同应激状态下的大鼠一定时间内糖水摄取量、排便颗粒、直肠扩张时腹肌收缩次数的变化,对模型进行再评价。采用免疫组织化学方法检测各组大鼠额叶、海马、脊髓背角、结肠c-fos水平;免疫组织化学方法检测各组大鼠结肠嗜铬细胞(EC),甲苯胺蓝染色检测大鼠结肠肥大细胞(MC)及活化肥大细胞百分比(活化MC%);免疫组织双染方法检测各组大鼠结肠酪氨酸羟化酶(TH)及其活化程度;Western Blot方法检测各组大鼠结肠TH蛋白水平;实时RT-PCR方法检测各组大鼠结肠和人类结肠粘膜TH-mRNA和c-fos-mRNA水平;MALDI-TOF-MS技术检测各组大鼠结肠蛋白指纹图谱。结果:1.联合应激大鼠额叶、海马、脊髓背角Fos蛋白明显高于对照组(P=0.026,0.002,0.013);结肠Fos蛋白略高于对照组(P=0.205);而与慢性应激大鼠相近(P=0.733)。急性应激大鼠中枢(额叶、海马、脊髓背角)与结肠c-fos-mRNA、Fos蛋白与对照组亦无显著差异(P＞0.05)。2、联合应激可使大鼠结肠分泌5-HT的EC和MC增多,MC活化脱颗粒,5-HT合成增加(P＜0.05),5-HT含量亦增多,但对结肠5-HT代谢的其他指标无显著影响。急性应激组结肠MC无明显变化,而脱颗粒MC%明显升高,EC较对照组增多(P＞0.05);慢性应激组结肠EC、MC和脱颗粒MC%增多;结肠TPH1 mRNA明显增高,结肠5-HT阳性细胞数增多(P＜0.05)。3、联合应激大鼠结肠NE含量显著增高,TH蛋白、TH-mRNA有升高趋势(P=0.590,0.363);急性应激组结肠NE无明显变化,TH蛋白、TH-mRNA有增高趋势(P＞0.05);慢性应激大鼠结肠NE含量有减低趋势,TH蛋白、TH-mRNA无明显变化(P＞0.05)。4、结肠5—HT系统和TH间相关系数无统计学意义(P＞0.05)。5、腹泻型IBS患者结肠粘膜TH和c-fos mRNA表达高于对照组(P=0.466,0.110),便秘型IBS患者结肠粘膜TH—mRNA低于IBS-D,而与对照组无明显差异(P＞0.05)。6、联合应激大鼠与对照组问有明显差异(P＜0.05)的蛋白质/肽质量差异峰18个,与慢性应激大鼠间有10个,与急性应激大鼠间有1个,全部为联合应激组上调。各应激组之间的差异蛋白有一定重复性。结论:1、联合应激大鼠脑-肠轴各层面尤其是脊髓背角、海马Fos蛋白显著升高,而单纯急性应激模型Fos蛋白无明显升高,提示慢急性联合应激模型不同于单纯急性束缚应激模型,是可以部分模拟脑-肠交互作用的动物模型,为从分子水平研究IBS脑-肠交互作用提供平台。2、动物与人结肠标本研究显示:慢性应激调动机体5-HT系统和NE系统处于活跃状态,在此状态下,5-HT系统和NE系统对新的急性应激的反应较正常增强,5-HT和NE总量增多,前者通过抑制5-HT降解,后者则通过NE合成增加实现。在机体对慢急性联合应激的反应中,肠道局部以5-HT系统作用为主导,NE系统则起辅助作用。提示精神因素对其它刺激有一定放大作用。3、M/Z为4011.172、29150.9、15213.25Da的蛋白/肽峰,在各组间均存在差异。应激越强,蛋白表达量越高。这三个蛋白质/肽峰可能最有意义。更多还原

【Abstract】 Background:Irritable bowel syndrome(IBS) is a common functional bowel disease.The etiology and pathogenesis are incompletely understood.Its pathophysiological basis is altered gastrointestinal motility and visceral hypersensitivity,usually accompanied with psychiatric disorder.Monoamine neurotransmitters in enteric nervous system,including 5-hydroxytrypatmine (5-HT) and norepinephrine(NE),play an important role in gastrointestinal sensation,motility and secretion.Abnormalities of serotonin system and sympathetic nervous system are related to the pathophysiology of IBS.An gut-brain interaction animal model of IBS,also called Chronic & Acute stress model(CAS),has been proved well repetitive.Major characteristics of IBS,such as altered colon motility,visceral hypersensitivity,and psychiatric disorder are existed in this model.We used CAS model to study the following plans on the basis of the former study about 5-HT and NE in the IBS rat model.The whole study includes three parts:Aims:1.Detecting c-fos contents in the colon,cornu dorsale,hippocampus and frontal]obe of rats,to evaluate the CAS model further.2.Perfecting and further investigating the changes and possible role of 5-HT system and NE system in IBS.3.Observing the overall view of protein of colon of brain-gut interaction IBS model utilizing proteomics,so as to study whether there are abnormalities in protein levels in IBS.Materials and Methods:Health male adult Wistar rats were selected and divided into four groups: control,acute stress(AS),chronic stress(CS) and Chronic & Acute stress(CAS).The consumption of sucrose solutions,the fecal pellets, frequency of abdominal muscle contraction by colorectal balloon distention (CRD) of rats after different stresses were observed to reevaluate colon motility,visceral sensation and behavior changes of model.Immunohistochemical method was used to determine enterochromaffin cells and toludine blue staining was used to measure mast cells in colon of rats. Double immunohistochemical method was used to detect TH and the degree of its activation.The change of TH protein level in the colon of rats was examed by Western Blot.The changes of TH mRNA and c-fos mRNA expression in the distal colon of rats and the colon mucosa of human were detected by Real time RT-PCR. MALDI-TOF-MS was used to measure the protein fingerprint in colon of rats.Results:1.Compared with the control,the levels of Fos protein in frontal lobe, hippocampus and cornu dorsale of CAS rat were significantly elevated (P=0.026,0.002,0.013),but its level in colon was slightly increased(P=0.205); and similar results were seen in those of CS rats.Fos protein contents in frontal lobe,hippocampus,cornu dorsale and colon of AS were no different compared with the control(P＞0.05).2.CAS led to increased amount of EC,MC and percentage of activated MC (activated MC%)(P＜0.05).The amount of MC and EC in the colon of AS was no different from that of control group(P＞0.05),but AS resulted in increased activated MC%(P＞0.05).The amount of EC,MC and activated MC%increased significantly in the colon of CS(P＜0.05).3.The content of NE in the colon of CAS rats was higher than that of the control(P＞0.05),and TH protein and TH mRNA increased without statistical significance compared to those of the control(P=0.590,0.363).Compared to the control group,the content of NE in the colon of AS was no different,and TH protein and TH mRNA had the increased tendency(P＞0.05).The NE level in the colon of CS rats had decreased tendency,and TH protein and TH mRNA were no different(P＞0.05).4.The correlation coefficient between colon 5-HT systems and TH was no statistically significant(P＞0.05).5.TH and c-fos mRNA levels in the colon mucosa of diarrhea-predominant IBS(IBS-D) patients were slightly higher than those of normal control (P=0.466,0.110).TH mRNA in the colon mucosa of constipation-predominant IBS(IBS-C) patients was lower than that of IBS-D patients,but no different from that of normal control(p＞0.05).6.As far as protein/peptide quality different peak was concerned,CAS rats have 18 different peaks compared with the control rats,10 peaks compared with CS rats,and 1 peak compared with AS rats.The protein level in the colon of CAS rats was highest.The different proteins among CAS,CS,AS and the control groups were repeated.7.Compared with the control group,the number of fecal pellets and frequency of abdominal contraction induced by CRD increased,and the amount of weight gain and consumption of sucrose solutions decreased in CAS(P＜0.05).Conclusions:1.CAS led to significantly increased Fos protein in parts of brain-gut axis(especially cornu dorsale and hippocampus),while AS couldn’t activate the express of c-fos.This demonstrates CAS model is different from single AS model and is a good one that can simulate brain-gut interaction partly.2.Stimulation such as chronic stress induced 5-HT system and NE system in active state.Under this active state,5-HT system and NE system show stronger reaction to new and acute stress than normal,and this resulted in increased 5-HT and NE contents because CAS can restrain degradation of 5-HT and increase synthesis of NE.In the response to CAS,5-HT system plays a predominant role and NE system plays an adjunctive role in the intestinal tract. It concluded that psychiatric stress can amplify the action other kinds of stimuli.3.The proteins/peptides whose M/Z are 4011.172、29150.9、15213.25Da were different among all groups.The stronger the stress is,the higher protein level is.The three proteins/peptides probably were the most significant.更多还原