【作者】 王军；

【导师】 房殿春；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 胃食管反流病(Gastroesophageal reflux disease,GERD)包括非糜烂性反流病(non-erosive esophageal reflux disease,NERD)、反流性食管炎(reflux esophagitis,RE)和Barrett食管(Barrett Esophagus ,BE),是危害人们健康的常见疾病,严重者可发展成为食管腺癌。因GERD及其并发Barrett食管和食管腺癌的发病机制尚不明确,从一定程度上影响了对它们的诊断和治疗。Claudin蛋白家族是近年来被发现的穿膜蛋白,是紧密连接最重要的组成部分,维持着紧密连接的特有的屏障功能和栅栏功能。在正常食管粘膜,claudin 3和claudin 4蛋白基本不表达;而在肠化的BE食管上皮细胞和食管腺癌组织中,二者表达明显增高。NERD和RE是我国GERD最主要的2个亚型,然而迄今对claudin 3、4在NERD和RE患者食管粘膜中的表达变化规律及其影响因素尚不清楚,在BE表达增高的信号机制也未见报导。为此,本课题重点进行4方面的研究:1.采用RT-PCR、western blot和免疫组织化学等方法,分别对claudin 3、4在正常食管、NERD、RE和BE粘膜上皮的表达情况进行检测,以此初步探讨其在GERD临床诊断和治疗中的意义;2.采用便携式24h pH监测仪及便携式24 h胆红素监测仪,对NERD和RE患者食管内pH及胆红素实行24h双监测,并用RT-PCR法检测被测食管粘膜内claudin 4 mRNA的表达变化,以此探讨胃酸和胆汁酸反流与食管粘膜claudin 4表达的联系;3.采用MTT法检测胃酸、胆汁酸和TNF-α对小肠癌上皮细胞株活力的影响,以此选定合适的实验条件,再分别采用实时-PCR和激光共聚焦的方法,观察胃酸、胆汁酸和TNF-α对小肠癌上皮细胞株claudin 4mRNA和蛋白表达水平及细胞内分布的直接影响。4.采用p38激酶特异性拮抗剂(SB203580),通过阻断p38激酶活化前后,western blot法检测胃酸、胆汁酸诱导的claudin 4蛋白表达的变化,以此研究p38激酶信号在胃酸和胆汁酸诱导的小肠上皮细胞claudin 4表达变化中的作用。主要结果如下:1.对80例GERD患者食管粘膜进行检测,发现claudin 3在正常食管,在NERD和RE的上皮,无论是mRNA,还是蛋白水平,不表达或微弱局灶表达;在肠化型BE上皮,claudin 3明显高表达。claudin 4在正常食管上皮微弱或不表达,而在NERD、RE和BE表达在mRNA和蛋白水平逐渐增强。免疫组化还发现claudin 3、4的着色在NERD和RE粘膜上皮主要在细胞膜;对于BE,二者不仅胞膜,胞浆内也出现阳性着色。2.对其中的48例GERD患者进行食管胃酸和胆红素监测,结果发现NERD组以无反流为主, RE组以混合反流为主,二者构成比相差显著;NERD组和RE组在pH<4总时间%、pH<4卧位时间%、酸反流次数、DeMeester积分、abs>0.14卧位时间%和胆汁反流次数等6个参数上存在明显差别(P<0.05);RE组食管粘膜claudin 4 mRNA的含量显著高于NERD。单独酸反流、单独胆汁反流和混合反流组claudin 4 mRNA均显著高于无反流组(P<0.05),单独酸反流和单独胆汁反流之间相差不显著(P>0.005),混合反流组含量最高,显著高于其他各组(P<0.05);GERD患者食管粘膜claudin 4 mRNA的含量与pH<4卧位时间%、酸反流次数,DeMeester积分,以及abs>0.14卧位时间%和胆汁反流次数等5个参数明显相关。3. MTT法结果显示pH选取的范围在pH 5.5～7.4,鹅脱氧胆酸(CDCA)50μM～200μM对小肠癌细胞株活力无明显毒性,可以作为合适的研究条件。激光共聚焦结果显示CDCA或盐酸随着作用时间增加和浓度增大, claudin 4 mRNA和蛋白的表达逐步增加;在刺激因素下蛋白表达的增强在HIC细胞内弥漫分布;在表达量和升高的速度上胆汁酸强于胃酸的作用;胆汁酸在pH 6.0的酸性培养基内的作用更高于单独胆汁酸的作用。TNF-α不同时间刺激后,claudin 4 mRNA和蛋白表达无显著变化。4.盐酸、CDCA单独或者混合刺激HIC细胞均可引起p38激酶活化,混合刺激活化p38激酶的程度明显高于单独刺激,同时伴有claudin 4蛋白表达的增高,使用p38激酶信号途径特异性拮抗剂SB203580,可以抑制p38激酶活化,同时可以抑制盐酸或胆汁酸单独刺激引起的claudin 4表达的增高,也可以部分抑制盐酸和胆汁酸混合刺激引起的表达增高。由上述结果得出以下结论:1.提示claudin 4表达的水平和粘膜病变程度密切相关,它表达增高有可能是食管粘膜上皮细胞紧密连接受损伤的标志,易引起粘膜损害的进一步加重;claudin 3是BE理想的诊断标志物和治疗的新靶点,claudin 3作为BE食管标志物的特异性要高于claudin 4。2.卧位酸、胆汁反流时间延长,反流的次数增多更易诱发RE,联合反流的效果更加明显。胆汁酸可能和胃酸一样,引起claudin 4的高表达,因而也是独立的食管粘膜损伤因子;胃酸、胆汁酸协同作用,更有致病性。卧位酸、胆汁反流时间和反流次数的增加是引起食管claudin 4表达增高的重要因素。3.胆汁酸或/和胃酸可能从基因转录和蛋白表达水平上直接影响barrett食管claudin4的表达,并具有时间和浓度效应。胆汁酸的作用强于胃酸,混合反流效果更加明显。尽管TNF-α未影响claudin 4的表达,但它在barrett食管的作用还需要进一步研究。4.胃酸、胆汁酸可能直接引起barrett食管的p38激酶活化,进而正向调节claudin 4蛋白的表达,除了p38激酶信号外,可能还存有其他调控机制参与claudin 4蛋白表达的增高。更多还原

【Abstract】 Gastro-esophageal reflux disease (GERD), including non-erosive esophageal reflux disease (NERD), reflux esophagitis (RE) and Barrett Esophagus (BE) is a prevalent chronic disease, and could develop into lethal esophageal adenocarcinoma. As the pathogenesis remains unclear, which , to some extent, affects its diagnosis and therapy. Claudins (CLDNs), the recently identified multigene transmembrane protein family, are thought to be the major constituents of tight junctions (TJ), the most apical components of cell sealing complexes responsible for cell–cell adhesion, cell polarity, and control of paracellular ion transport. The expression of claudin 3 and 4 protein were not observed in the esophageal mucosal layers, but signifcantly increased in the epithelial cell of BE and tissue of esophageal adenocarcinoma. Although NERD and RE are the two main sub-types in our country, the changes of their expression and their impacting factors remain unclear, as well as the signal mechanism. Therefore, the following experiments were performed in this study.1. By the methods of RT-PCR, western blot and immunohistrochemistry, the expression of claudin 3 and 4 in normal esophageal, NERD, RE and BE were detected , by which we could explore their significance in the diagnosis and therapy of GERD.2. With the simultaneous ambulatory 24 hour oesophageal pH monitoring meter and 24h bilirubin monitor meter, pH value and bilirubin concentration in lumina of NERD and RE patients were mornitored. In the meanwhile, by the methods of RT-PCR, the mRNA expression of claudin 4 in the mucous membrane of esophageal was detected.3. By the method of MTT, the proliferation of human small intestine carcinoma cell line(HIC) was analyzed. The effects of hydrochloric acid, chenodeoxycholic acid(CDCA) and TNF-αon the cell line were evalued. Based on the above results, the suitable experimental parameters were chosen. The mRNA and protein expression of were investigated by the methods of real time PCR and Laser scanning confocal microscop(LSCM). By which, the effects of the injury factors such as gastric acid, CDCA and TNF-αon claudin 4 expression were investigated.4. With the drug tool of p38 kinase specificity antagonist--SB203580, the expression of claudin 4 protein was detected before and after p38 kinase signal activated, which could explore the role of p38 kinase signal in the gastric and bile acid induced claudin 4 protein expression.The main results were as followings:1. Results of 80 cases of GERD patients showed that claudin 3 did not or mildly express in the mucous membrane of normal esophagus, NERD and RE, but increased signifcantly in BE group.The mRNA and protein expression of claudin 4 was weak in normal esophageal group. While in NERD, RE and BE group, they increased gradually. The results of immunohistochemistry showed that both claudin 3 and claudin 4 positive stain was mainly in the cellular membrane of epithelial cells in NERD and RE group, while in the BE group, the positive stain both in the cellular membrane and cytoplasm.2. Results of monitoring gastric acid and bilirubin in 48 cases of GERD patients showed that there were no reflux in most NERD patients, while in RE group was mainly mixture reflux, and the percent of constitutent differed significantly. Significant differences were also observed between NERD and RE groups in the six parameters such as percent of total time the pH<4, percent supine time pH<4, No. of acid reflux episodes, DeMeester score, percent supine time the bilirubin absorbance level≥0.14 and number of bilirubin reflux episodes (p<0.05). The mRNA expression of claudin 4 in RE group was significantly higher than that in NERD group (p<0.05). The mRNA expressions of claudin 4 in the gastric acid reflux alone, bilirubin reflux alone and mixture reflux group were significantly higher than that in no reflux group, respectively (p<0.05), no difference between in the gastric acid reflux alone and in bilirubin reflux alone group. The expression was highest in the mixture reflux group, significantly higher than that in other group (p<0.05). The mRNA expression of claudin 4 in GERD patients was in correlation to percent supine time pH<4,No. of acid reflux episodes,DeMeester score, percent supine time the bilirubin absorbance level≥0.14 and number of bilirubin reflux episodes. 3. Results of MTT experiments showed that pH value ranged 5.5～7.4 and concertration of CDCA ranged between 50μM～200μM had no significant toxicity on the proliferation of HIC cells, which could be chosen as the suitable experimental parameters. Results of confocal showed that the mRNA and protein expression of claudin 4 increased with the time and concentrations of CDCA and hydrochloric acid. The increasing expression of claudin 4 protein distributed diffusely in the HIC cells in the presence of stimulation. No difference was observed in the mRNA and protein expression of claudin 4 after stimulation of TNF-αin different stimulus duration.4. P38 kinase could be actived by either acid hydroc, CDCA alone or the two combination in the HIC cell line. And the amount actived by CDCA with acid hydroc was significant higher than that actived by acid hydroc or CDCA alone. Meanwhile, the expression of claudin 4 inreased significantly. SB203580, as the p38 kinase signal specificity antagonist, inhibited the activation of p38 kinase and the expressions of claudin 4 induced by hydrochloric acid and bile acid alone or both.We could draw conclusions from the above results.1.The level of claudin 4 expression was closely associated with the severity of pathological changes in the mucous membranes. The high level of claudin 4 expression could be the new marker of the injury of tight conjunction in the mucous membrane. Be the new targets of diagnosis and therapy of BE, The specificity of claudin 4 in the diagnosing BE was lower than that of claudin 3.2. With the increasing of supine time and nubmer of reflux of acid and bilirubin ,RE would be more easily induced. And the effect of the combined stimulation was more obvious. Bile acid alone could induce high expression of claudin 4, therefore, which could be the independent factor of injurying esophageal mucous membrane. The synergism effect of gastric acid and bile acid on the claudin 4 expression and pathopoiesis was more obvious.3. With time and dose-dependent, bile acid and/or gastric acid could affect the expression of claudin 4 from the level of gene transcription and protein expression. And the effect of bile acid was more stronger than that of gastric acid, the combined reflux the strongest. Although TNF-αhad no effect on the expression of claudin 4, its role on the pathegensis of barrett esophageal need be further studied.4. Gastric acid and bile acid could induce the activation of p38 kinase of BE directly and then positively regulate the expression of claudin 4. Besides of p38 kinase signal, there would be other regulatory mechanisms involved in the increasing expression of claudin 4 protein.更多还原