【作者】 陈卫；

【导师】 刘玮丽；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2018， 硕士

【摘要】 背景炎症性肠病(inflammatory bowel disease,IBD)是一类多病因引起的、异常免疫介导的肠道慢性及复发性炎症,具有慢性迁延、反复发作、难以治愈的特点,其主要疾病类型是溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn disease,CD),临床表现以腹痛、腹泻、黏液脓血便、体重减轻为主。其中溃疡性结肠炎是目前公认的结肠癌前病变之一,年癌变率约3‰[1],相比于散发的结肠癌,UC相关的结肠癌更易在结肠中多个区域连续分布,肿瘤分期也更高[2]。其发病率在我国呈逐年递增趋势。许多人类和动物的研究表明UC是环境因素作用于遗传易感性患者导致对自身肠道菌群失去耐受的结果[3]。溃疡性结肠炎的特征在于结肠上皮细胞过度增殖,信号传导通路失调,肠粘膜T细胞过度活化,促炎细胞因子的产生增加,结肠间隙的白细胞浸润增加[4]。近年研究表明肠道免疫功能紊乱是导致溃疡性结肠炎慢性炎症发生的重要因素之一。溃疡性结肠炎患者肠道Th2细胞异常活化[3];炎症细胞(包括中性粒细胞和单核细胞)浸润肠壁并释放的大量炎症介质(如TNF-α、IL-1、IL-4、IFN-y等),直接或间接调节T淋巴细胞功能[5];细胞因子失调与Th1/Th2失衡互为因果,加剧炎症性肠病的免疫病理过程,诱导肠上皮细胞癌变发生。因此调节异常炎症介质的释放是治疗溃疡性结肠炎的重要靶向之一。雷帕酶素靶蛋白 mTOR(mammalian(or mechanistic)target of rapamycin)是一种高度保守的丝氨酸-苏氨酸蛋白激酶,它可以调节蛋白质的合成,细胞生长和增殖,在肿瘤发生中也发挥至关重要的作用[6,7]。越来越多的证据表明mTOR在组织细胞对炎症介质(如肿瘤坏死因子TNF)的响应中起到重要的作用。PeairsA等[8,9]发现绿茶中的活性抗炎物质通过作用PI3KK/Akt/mTOR通路抑制mTOR活性;mTOR活性降低的同时伴随LPS/IFN-y联合刺激下的MRL/lpr小鼠系膜细胞的COX-2、IL-6等炎症介质的释放的大幅度减少。Gabriela等人[100]运用“多柔比星-二甲双胍-草氨酸钠”三联疗法治疗结直肠癌小鼠模型,结果显示可以显著降低肿瘤的数量和大小,还可以减少促炎细胞因子的产生。而这种疗法主要是通过靶向作用mTOR信号通路和乳酸脱氢酶A来发挥其抗肿瘤及抗炎的作用。rmTOR还可以通过调节巨噬细胞或树突状细胞内源性免疫反应,控制炎症介质释放,因此在感染性或自身免疫性疾病中均有重要作用[11]。雷帕霉素作为mTOR抑制剂,开始主要用于器官移植病人的免疫排斥反应,最近研究发现其对肾癌、肺癌、结肠癌均有治疗作用[12-14]。有研究报道mTOR抑制剂用于治疗中-重度CD患者,其疗效与硫唑嘌呤相当[15]。另一项研究显示用西罗莫司(雷帕霉素)作为辅助免疫抑制剂治疗儿童难治性炎症性肠病可使患儿获得临床缓解或者临床疗效[16]。但有关mTOR抑制剂治疗UC的研究少见。本文将研究mTOR在溃疡性结肠炎患者中的表达,并通过溃疡性结肠炎动物模型来进一步验证mTOR抑制剂对溃疡性结肠炎的作用,探讨溃疡性结肠炎发展分子机制,为溃疡性结肠炎的治疗提供新思路,为老药新用提供理论依据。方法1.临床溃疡性结肠炎病例的收集及mTOR检测:利用已建立的炎症性肠病数据库,调取溃疡性结肠炎患者相关信息及其肠镜检查石蜡标本、切片,ABC法免疫组化检测不同阶段溃疡性结肠炎患者组织标本中mTOR表达水平。2.通过溃疡性结肠炎动物模型研究mTOR抑制剂对溃疡性结肠炎发展的影响:建立小鼠溃疡性结肠炎模型,同时予mTOR抑制剂干预溃疡性结肠炎小鼠,检测模型中mTOR表达情况,观察组织病理学改变。结果1.通过免疫组化方法检测发现:mTOR在活动期UC患者中的表达较缓解期升高;且在活动期UC患者(轻度、中度、重度)中,随着疾病活动程度加重,mTOR表达进行性增高。2.我们用2%葡聚糖硫酸钠(dextransufatesodium,DSS,30-40kDa)给小鼠饮用7天,继以普通饮用水14天为一周期,循环两周期建立溃疡性结肠炎动物模型,同时给予mTOR抑制剂依维莫司干预。在DSS诱导的小鼠溃疡性结肠炎动物模型中,1)mTOR抑制剂干预可预防DSS诱导的结肠缩短。2)组织学分析显示:mTOR抑制剂减轻了 DSS导致的结肠组织水肿,显著减少结肠粘膜的炎性细胞浸润,并且对DSS诱导的隐窝破坏有保护作用。3)干预组小鼠mTOR表达低于对照组。结论1.mTOR在活动期UC患者中表达较缓解期升高;且活动性UC患者中,随着疾病活动程度加重,mTOR表达进行性增高。2.mTOR抑制剂对小鼠结肠炎模型进行干预可减少mTOR表达,改善结肠炎。更多还原

【Abstract】 BackgroundInflammatory bowel disease(IBD)is a chronic non-specific gastrointestinal inflammatory disease,consists of ulcerative colitis(UC)and Crohn’s disease(CD).And ulcerative colitis is one of the recognized precancerous lesions,The overall incidence rate of colorectal cancer in UC patient was 3/1000 person years duration.Its incidence rate has been increasing year by year in China.Ulcerative colitis is an autoimmune-inflammatory disease characterized by increased proliferation of colonic epithelial cells,dysregulation of signal transduction pathways,elevated mucosal T cell activation,increased production of proinflammatory cytokines,and enhanced leukocyte infiltration into colonic interstitium.Mammalian target of rapamycin(mTOR)is a serine-threonine protein kinase that regulates protein synthesis,cell growth,and cell proliferation in response to growth factors and nutrients.More recently,accumulating evidence causally links increased mTOR activity to heightened inflammatory responses.The inhibitor of mTOR——rapamycin,was firstly used for immune rejection in organ transplant patients.Walter Reinisch and his colleagues reported that the safety and tolerability of everolimus(6 mg/day)in patients with active CD were comparable to azathioprine.However,it is rare to treat UC patients with mTOR inhibitors.We will observe whether the expression of mTOR is related to the activity of ulcerative colitis firstly;then,we will observe the role of mTOR on the development of ulcerative colitis in mice model.This research is to investigate the association between mTOR and ulcerative colitis.MethodsThe samples of 55 cases of UC tissues were collected.By using immunohistochemistty,the expression of mTOR was detected.Then,we established the mice model of ulcerative colitis and detected the expression of mTOR in the model.Then the mice was treated with the everolimus to probe the efficacy of inhibitor of mTOR.Chronic colitis was induced in mice by giving 2%DSS(molecular mass 30-40 kDa)for 1 week then water for 2 weeks for two cycles.For each mouse,weight and rectal bleeding were determined every day following the introduction of DSS.To probe the efficacy of everolimus,a group of ten mice were given daily gastric perfusion of 3 mg/kg everolimus at the beginning of the experiment.Another group of ten mice were given daily gastric perfusion of the same volume of water.At the end of the experiment,the mice were killed and colon tissues were collected.Results:The results of IHC indicates mTOR is overexpressed in the patients with UC.And in different stages of ulcerative colitis,the expression of mTOR is progressive.In the dextran sulfate sodium(DSS)model of colitis,1)macroscopic colon observations demonstrated that everolimus significantly reversed DSS-induced shortening of the colon;2)histological analyses of colonic tissues revealed that everolimus distinctly attenuated DSS induced edema,prominently diminished the leukocyte infiltration in the colonic mucosa,and resulted in protection against DSS-induced crypt damage.Conclusion:Collectively,these results indicate that mTOR is overexpressed in UC.Everolimus preventive treatment of established colitis in mice ameliorated the colitis.更多还原