【作者】 房渝；

【导师】 胡建国；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景和目的：食管上皮屏障功能是食管粘膜对抗胃食管反流的重要防御机制。胃食管反流可诱导食管上皮屏障功能的损伤,食管粘膜对特定离子以及大分子通透性的增强以及上皮细胞间隙的增宽。因此,理解反流诱导的食管粘膜屏障功能损伤的机制对寻求胃食管反流病的有效干预措施具有重要的临床意义。Barrett食管是胃食管反流病的继发病变之一,但胃食管反流进展为Barrett食管的分子机制至今还不清楚。本研究的目的1)探讨NFκB介导的炎症在胃食管反流诱导食管粘膜屏障功能损伤中的作用；2)探讨胃食管反流病进展为Barrett食管的分子机制。研究方法：建立手术诱导的胃反流,十二指肠反流和胃十二指肠混合反流的小鼠模型.使用mini-Ussing chamber检测各反流食管粘膜跨上皮电阻抗值,利用电镜观察并比较各型反流食管粘膜上皮间隙的改变。应用基因芯片技术检测各型反流食管粘膜上皮内特异的基因表达谱,再利用生物信息学分析策略(例如GSEA和SAM)筛选出表达上调或者下调的转录因子,激活的经典信号传导通路以及具有相似功能的基因簇,同时也筛选出基因组内所有表达上调或者下调的基因。采用Real-time PCR,免疫印迹,ELISA和免疫组化方法检测筛选基因的表达水平。使用特殊组织化学染色来显示各型反流食管粘膜内炎症细胞的分布,并对炎症细胞的数量进行统计比较。分别给予十二指肠反流和混合反流模型小鼠腹腔注射NFκB抑制剂Bay11-7085(20mg/kg/day),观察食管粘膜跨上皮电阻抗以及炎症因子水平的改变。研究结果：与正常小鼠食管粘膜上皮比较,十二指肠反流和混合反流食管粘膜的跨上皮电阻抗明显下降,但胃反流粘膜并未有明显改变。电镜观察到十二指肠反流和混合反流食管粘膜上皮细胞间隙明显增宽,而胃反流食管粘膜间隙并未明显改变。GSEA分析表明,在各型反流食管粘膜中有大量炎症相关基因簇和经典信号传导通路被激活,同时在十二指肠反流和混合反流食管粘膜上皮中可见NFκB表达上调。通过将SAM数据与已知NFκB下游基因、紧密连接蛋白基因以及Barrett食管相关基因进行比对,筛选出一系列上调或者下调的NFκB下游基因、紧密连接蛋白基因以及Barrett食管相关基因。Real-time PCR显示在十二指肠反流和混合反流食管粘膜上皮中紧密连接蛋白Cldnl, Cldn4, Cldn10和Cldn23的表达均下调,同时Cldn7的表达上调。免疫印迹和免疫组化显示在十二指肠反流和混合反流食管粘膜中Cldnl和Cldn4蛋白表达水平明显下调并且在食管上皮内的分布发生了明显改变。免疫组化显示NFκB亚基p50和p65, NFκB下游基因MMP3和MMP9以及Cdx2在十二指肠反流和混合反流食管粘膜上皮中的表达均明显上调。十二指肠反流和混合反流食管粘膜中炎症因子水平以及炎症细胞计数均明显增加。NFκB抑制剂Bay11-7085可以明显减弱反流对食管屏障功能的损害并且抑制反流诱导食管粘膜炎症因子水平的上调。研究结论：1)胃食管反流通过NFκB介导的炎症损伤小鼠食管上皮屏障功能；2)胃食管反流通过激活Barrett相关基因的表达从而导致Barrett食管的发生。更多还原

【Abstract】 Background and Aim:The barrier function of esophageal epithelium is a major defense against gastroesophageal reflux. Previous studies have shown that reflux damage is reflected by increased permeability of esophageal mucosa for ions and macromolecules and dilated intercellular space in the esophageal epithelium. In order to develop novel therapies, it is critical to understand how contact with a refluxates attenuates esophageal barrier function. Barrett metaplasia usually occurs as a consequence of gastroesophageal reflux disease, but the exact molecular mechanism of gastroesophageal reflux progressing into Barrett esophagus remains poorly understood. This study aims1) investigating the role of NFκB-mediated inflammation in attenuated esophageal barrier function due to gastroesophageal reflux;2) investigating the molecular mechanism of gastroesophageal reflux progressing into Barrett esophagus.Methods:In this study, gastric, duodenal and mixed reflux models were developed in mice through surgical performance. Transepithelial electrical resistance (TEER) was measured by mini-Ussing chamber for different reflux models, while dilated intercellular space (DIS) observed with electrical microscope. Mouse esophageal epithelium is analyzed by gene microarray and statistical strategies (i.e.,Gene Set Enrichment Analysis and Significance Analysis of Microarray), which screened out activated transcription factors, canonical signaling pathways and gene ontology, as well as up-or down-regulated genes in mouse genome. Genes screened were confirmed by gene expression assays (i.e.,Real-time PCR, Western Blotting, immunohistochemical staining and ELISA). Distribution of inflammatory cells in esophageal mucosa of different reflux models was shown by specific histochemical staining, while semi-quantification of inflammatory cells was caculated. Upon mice treated with NFκB inhibitor Bay11-7085(20mg/kg/day, i.p.), TEER and cytokines were measured in esophageal mucosa of duodenal and mixed reflux models.Results:A decrease of TEER and DIS was observed in esophageal mucosa of duodenal and mixed reflux models as compared with control, but not in that of gastric reflux model. GSEA showed the activation of inflammation-related gene sets and canonical signaling pathways in esophageal epithelium of different reflux models, and activation of NFκB due to duodenal and mixed reflux. SAM revealed up or down-regulation of NFκB target genes, Barrett-related genes and tight junction genes. Real-time PCR confirmed down-regulation of Cldnl, Cldn4, Cldn10and Cldn23and up-regulation of Cldn7. Immunohistochemical staining showed up-regulation of NFκB p50/p65, NFκB target genes MMP3/MMP9and Cdx2in esophageal epithelium of mixed and duodenal reflux models. An increase of cytokines (i.e. IL1β, IL6and IL8) and cell counts (i.e. neutrophils, eosinophils and mast cells) was observed in esophageal mucosa of duodenal and mixed reflux models as compared with control. Treatment with an NFκB inhibitor, Bay11-7085counteracted the effects of duodenal and mixed reflux on TEER and cytokines.Conclusion:Our results suggested1) gastroesophageal reflux attenuates the barrier function of mouse esophageal epithelium through NFκB-mediated inflammation;2) Gastroesophageal reflux induces Barrett’s metaplasia in esophageal epithelium through activation of Barrett’s genes.Nineteen pictures,5tables and85references are included in this manuscript.更多还原