喉咽反流动物模型中喉部粘膜屏障的防御机制研究

发布时间：2018-03-10 10:22

本文选题：兔　切入点：胃食道反流　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分喉咽反流病兔动物模型的建立与评价目的：建立和评估喉咽反流兔动物模型并利用此模型研究下食道括约肌功能障碍和反回流性喉炎之间的关系。方法：12只健康新西兰大白兔(2.5-3.5公斤)分成两组。8只行下食道括约肌全切除术以诱导反流。其余的四只行假手术作为对照组。所有动物术前行喉镜检查和24小时食管内pH值检测,术后2周和8周重复以上检查,然后处死所有动物,以获得组织学的结果。结果：下食道括约肌全切除术使实验组动物术后的反流指数(reflux index, R),最长反流持续时间(duration of the longest reflux episode, DLRE)和24小时内总反流次数显著增加。实验组术前和术后2周反流喉镜评分(reflux finding score, RFS)无显着性差异(P=0.11)。但实验组术前和术后8周RFS由4.6+0.9上升到8.3+3.6,有显着性差异(P=0.02)。实验组与对照组相比,粘膜下腺体增生和炎症反应有显著性差异。结论：本研究结果显示,在兔动物模型中慢性食管下段括约肌功能障碍可导致反流性喉炎。第二部分喉咽反流病兔动物模型食道及声带粘膜细胞间隙的研究目的：本研究中,我们利用反流动物模型研究了兔喉和食道上皮在透射电镜下的组织学变化,从而揭示喉咽反流(laryngopharyngeal reflux, LPR)和胃食管反流(gastroesophageal reflux, GER)的关系以及扩张的细胞间隙(dilated intercellular space,DIS)可否作为LPR形态标志。方法：8只新西兰大白兔分为2组。4只兔子行下食道括约肌全切除术以诱导反流。余下4只行假手术,作为对照组。所有动物术后12周后处死以获得组织学及电镜结果。结果：实验组和对照组的食道和喉组织学结果有显著性差异(P=0.041和0.014)。与对照组相比,实验组食道和喉上皮细胞间隙(intercellular space,IS)有显著性差异(P0.001)。结论：本实验的结果提示LPR和GER有相似的发病机制,而且DIS可作为兔LPR模型的形态学指标。与对照组相比,实验组食道和喉上皮细胞间隙(intercellular space,IS)有显著性差异(P0.001)。第三部分紧密连接蛋白Claudin-3和Occludin在反流动物模型中的表达目的：建立和评估喉咽反流大鼠动物模型并利用此模型研究喉内claudin-3和occludin表达和喉咽反流之间的关系。方法：8只健康大白鼠(250-300 g)分成两组。4只行下食道括约肌全切除术以诱导反流。其余的四只行假手术作为对照组。所有动物术后12周处死,以获得组织学的结果。应用Western blot和免疫荧光检测食道及喉粘膜样本中claudin-3和occludin的表达。结果：实验组食道和喉内粘膜淋巴细胞浸润有显著增加(P=0.001和0.002)。实验组食道和喉内粘膜claudin-3表达水平明显低于对照组(P=0.045,0.037)；occludin表达无明显差异。结论：本实验结果提示在喉咽反流大鼠模型中,claudin-3表达的降低对于反流性喉炎有敏感的指示意义。
[Abstract]:Part I Establishment and evaluation of rabbit model of laryngopharyngeal reflux objective: to establish and evaluate the rabbit model of laryngopharyngeal reflux and to study the relationship between lower esophageal sphincter dysfunction and reflux laryngitis. 12 healthy New Zealand rabbits (2.5-3.5 kg) were divided into two groups: total resection of lower esophagus sphincter was performed to induce reflux in two groups. The other four rabbits underwent sham-operation as control group. All animals underwent laryngoscopy before operation and 24 hours esophageal pH test. Two and eight weeks after the operation, all the animals were killed. Results: total resection of lower esophageal sphincter significantly increased the reflux index (RV), duration of the longest reflux isodee (DLRED) and total reflux times in 24 hours. There was no significant difference in reflux finding score before and 2 weeks after operation in the test group (P < 0.01), but the RFS in the experimental group increased from 4.60.9 to 8.33.6 before and 8 weeks after operation, there was significant difference between the experimental group and the control group. There were significant differences in submucous gland hyperplasia and inflammatory response. Conclusion: the results of this study show that, Chronic lower esophageal sphincter dysfunction may lead to reflux laryngitis in rabbit models. We studied the histological changes of rabbit laryngeal and esophageal epithelium under transmission electron microscope (TEM) using a reflux animal model. To reveal the relationship between laryngopharyngeal reflux (LPRs) and gastroesophageal reflux (GERS), and whether dilated intercellular space DISs can be used as a morphological marker of LPR. Methods eight New Zealand white rabbits were divided into 2 groups with 4 rabbits with lower esophageal sphincter. Total resection to induce reflux. The remaining 4 were sham-operated. All animals were killed 12 weeks after operation to obtain histological and electron microscopic results. Results: there were significant differences in histological results of esophagus and larynx between experimental group and control group. Conclusion: the results of this study suggest that LPR and GER have similar pathogenesis, and DIS can be used as a morphological index of LPR model in rabbits. There was significant difference between the esophageal and laryngeal epithelial cells in the experimental group (P0.001). The third part of the tight junction protein Claudin-3 and Occludin expression in reflux animal model: to establish and evaluate the rat model of larynx reflux and use this model. To study the relationship between the expression of claudin-3 and occludin in larynx and pharynx reflux. Methods eight healthy rats were divided into two groups: total resection of lower esophageal sphincter to induce reflux. The animals were executed 12 weeks after operation. Western blot and immunofluorescence were used to detect the expression of claudin-3 and occludin in esophageal and laryngeal mucosa samples. Results: lymphocyte infiltration in esophagus and larynx of experimental group was significantly increased by P0. 001 and 0. 002. The expression of claudin-3 in the laryngeal mucosa was significantly lower than that in the control group. Conclusion: the decrease of the expression of claudin-3 in laryngopharyngeal reflux rat model has a sensitive indicative significance for reflux laryngitis.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R571;R-332

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