【作者】 郑秀丽；

【导师】 杨宇；

【作者基本信息】 成都中医药大学 ， 中医临床基础， 2013， 博士

【摘要】 目的：本实验通过建立“肺病”(慢性支气管炎)和“肠病”(溃疡性结肠炎)两种慢性非特异性炎症疾病动物模型,选取三个不同的时间点,同步观察肺与大肠微生态和MEK/ERK信号传导变化,探索肺与大肠病理传变的生物学基础。方法：以烟熏法复制肺病(慢性支气管炎)大鼠模型,分别在模型第20天、模型第50天和模型第70天三个时间点采取标本进行肺肠同步动态观察。以三硝基苯磺酸(TNBS)-乙醇相结合的方法诱导肠病(溃疡性结肠炎)大鼠模型,分别在模型第8天、第29天和第50天三个时间点采取标本进行肺肠同步动态观察。培养检测呼吸道和肠道的需氧细菌总数、厌氧细菌总数、肠杆菌、肠球菌、葡萄球菌、产气荚膜梭菌、双歧杆菌、乳酸杆菌；Western-Blot法检测肺组织和结肠组织匀浆中的MEK、p-MEK、ERK1/2、p-ERK1/2蛋白表达；ELISA法检测大鼠肺组织和结肠组织匀浆中的TNF-α受体、IL-1β受体和C-FOS含量。结果：1.呼吸道和肠道菌群：肺病大鼠各个时间点的肺病模型大鼠在肺部和肠道都分别出现菌群种类和数量不同程度的变化,模型第20天时,需氧菌总数在呼吸道和肠道同步增多,肠道微生物定植抗力(B/E值)显著下降,呼吸道与肠道的需氧菌总数和葡萄球菌中度相关(0.5≤r≤0.8)；模型第50天呼吸道与肠道的厌氧菌总数中度相关；模型第70天呼吸道与肠道的需氧菌总数和肠球菌中度相关。肠病大鼠出现了肠道菌群失调,益生菌数量减少,条件致病菌数量增多,同时,呼吸道菌群也出现部分菌群一定程度的相关变化。模型第8天时,需氧总数和葡萄球菌在呼吸道和肠道同步增多,厌氧总数和肠杆菌在肠道增多而在呼吸道减少。模型第29天时,需氧总数和葡萄球菌在在呼吸道和肠道同步减少,厌氧总数和肠杆菌在肠道减少而在呼吸道增多：模型第50天时,呼吸道和肠道的需氧菌总数、厌氧菌总数和葡萄球菌在呼吸道和肠道同步增多。2. MEK/ERK信号通路：肺病大鼠在模型第20天组、模型第50天组和模型第70天肺组织的p-ERK1/2含量均较空白组显著升高(P<0.05或P<0.01),肺组织的MEK/p-MEK、 ERK/p-ERK蛋白变化均比结肠组织的相应蛋白变化明显,肺与结肠的MEK/p-MEK和ERK/p-ERK蛋白变化具有较为一致的趋势。肠病大鼠在模型第8天结肠组织的ERK1/2和p-ERK1/2含量较空白组显著升高(P<0.01),第29天p-MEK含量显著升高(P<0.05)。与模型第8天比较,模型第29天ERK1/2和p-ERK1/2含量显著升高(P<0.01或P<0.05)；模型第50天p-MEK、ERK1/2和p-ERK1/2含量均显著降低(P<0.05)。肺组织仅在模型第29天与模型组第8天比较,肺组织的p-MEK含量显著升高(P<0.05)。结肠组织与肺组织的MEK/ERK呈现同步变化规律,或同步增多,或同步减少,或在肠增多而在肺减少,或在肠减少而在肺增多。3. TNF-α受体、IL-1β受体和C-FOS含量：肺病大鼠在肺病模型第70天,肺组织TNF-α受体和IL-1β受体含量显著减少(P<0.05),而结肠组织的TNF-α受体和IL-1β受体含量显著增加(P<0.05或P<0.01)。在模型第50天肺和结肠组织c-Fos含量均显著减少(P<0.05)。肠病大鼠在模型第8天结肠组织的TNF-α受体含量显著减少而肺组织的TNF-α受体含量显著增加(P<0.05)；在模型第8天和第29天结肠组织IL-1β受体含量显著增加而肺组织的IL-1β受体含量显著减少(P<0.05)：在模型第29天和模型第50天结肠组织c-Fos含量显著增加而肺组织的c-Fos含量显著减少(P<0.01)。结论：1.肺病大鼠可出现肠道菌群的改变,在“肺病及肠”病理传变过程中,其呼吸道微生态系统和肠道的微生态系统出现的失调,存在一定程度的动态相关性,肺与大肠在微生态方面的相互影响可能是“肺与大肠相表里”的内涵之一。2.肠病大鼠可出现呼吸道菌群的改变,在“肠病及肺”病理传变过程中,肠病大鼠呼吸道和肠道的部分菌群出现同步规律性变化。或同步增多,或同步减少。这说明微生态菌群的变化可能是“肠病及肺”的机制和表现形式之一。3.肺病大鼠的肺组织在MEK/ERK通路上变化具有一定的规律性,p-ERK1/2可能是慢性支气管炎在MEK/ERK通路上变化较为明显的指标,提示MEK/ERK通路可能是“肺病及肠”的生物学基础之一。4.肠病大鼠的肺组织和结肠组织的MEK/p-MEK和ERK/p-ERK蛋白变化具有较为一致的趋势,一定程度上体现了肺肠之间的同步变化。提示MEK/ERK通路可能是“肠病及肺”的生物学基础之一。5.肺病大鼠可出现肺肠TNF-α受体、IL-1β受体和c-Fos含量的同步动态变化,或在肺肠同步减少,或在肺减少而在肠增多。提示TNF-α受体、IL-1β受体和c-Fos可能是“肺病及肠”的部分物质基础。6.肠病大鼠可出现肺肠TNF-α受体、IL-1β受体和c-Fos含量的同步动态变化,或在肺肠同步减少,或在肠减少而在肺增多,或在肠增多而在肺减少。提示TNF-α受体、IL-1β受体和c-Fos可能是“肠病及肺”的部分物质基础。更多还原

【Abstract】 Objective:To establish pulmonary disease (chronic bronchitis) and bowel disease (ulcerative colitis) animal models respectively, which are both chronic non-specific inflammatory disease. Select three different points, synchronous observe the respiratory and intestinal micro flora, MEK/ERK signal pathway, and its upstream mediators of inflammation receptor TNF-alpha receptor, IL-1beta receptors and downstream effecter substances C-FOS both in lung and large intestine, to explore the biological basis of the mass change of the lung and large intestine in pathological state.Method:Use smoked method to copy pulmonary disease (chronic bronchitis) rat model, take samples of lung and intestinal synchronous dynamic observation respectively at model20th,50th and70th. Use TNBS combined ethanol method to copy bowel disease (ulcerative colitis) rat model, take samples of lung and intestinal synchronous dynamic observation respectively at model8th,29th and50th. Detect the total number of aerobic bacteria, total number of anaerobic bacteria, E. coli, enterococci, staphylococcus aureus, clostridium perfringens, bifidobacterium and lactobacillus both in respiratory and intestinal at the same time. Detect MEK, p-MEK, ERK1/2, p-ERK1/2protein expression in the homogenate of lung tissue and colon tissue by Western-Blot method. Use ELISA method to detect the content of TNF-alpha receptor, IL-1beta receptor and C-FOS in the homogenates of lung tissue and colon tissue.Results:1. Respiratory and intestinal flora:Pulmonary disease (CB) model rats have different degree changes of flora species and quantities at each time point in the respiratory and intestinal. At model20th, the total number of aerobic bacteria in the respiratory and intestinal increased simultaneously. Intestinal microbial colonization resistance (B/E) decreased significantly. The total number of aerobic bacteria and staphylococcus aureus of respiratory and intestinal are moderate correlated (0.5<r<0.8). At model50th, the total number of anaerobic bacteria in respiratory and intestinal are moderate correlated. At model70th, the total number of aerobic bacteria and enterococci in respiratory and intestinal are moderate correlated.Bowel disease (UC) model rats appear intestinal flora imbalance, the number of probiotics decreased but opportunistic pathogen increased. At the same time, part of the respiratory tract micro flora is also have related change in certain degree. At model8th, the total number of aerobic and staphylococcus aureus in the respiratory and intestinal increased simultaneously, the total number of anaerobic and enterobacteriaceae increased in the intestine but decreased in the respiratory tract. At model29th, the total number of aerobic and staphylococcus aureus in the respiratory and intestinal reduced simultaneously, the total number of anaerobic and enterobacteriaceae reduced in the intestinal but increase in the respiratory tract. At model50th, the total number of aerobic bacteria, the total number of anaerobic bacteria and staphylococcus aureus in the respiratory and intestinal increased simultaneously.2. MEK/ERK signaling pathway:In pulmonary disease (CB) model rats, the contents of p-ERK1/2were significantly higher than the blank group in the lung tissues at model20th,50th and70th(P<0.05or P<0.01).The changes of MEK/p-MEK and ERK/p-ERK in the lung tissue is significant than the colon tissue. The change trend of MEK/p-MEK and ERK/p-ERK protein in the lung and colon tissue is more consistent.In bowel disease (UC) model rats, the content of ERK1/2and p-ERK1/2were significantly higher than the blank group in the colon tissue at model8th(P<0.01). The content of p-MEK was significantly higher than the blank group in colon tissue at model29th (P＜0.05). The ERK1/2and p-ERK1/2at model29th were significantly higher than the8th (P<0.05or P<0.01). The content of p-MEK, ERK1/2and p-ERKl/2were significantly reduced at model50th(P<0.05). Only in the model29th, the content of p-MEK in the lung tissue was significantly higher than the8th (P<0.05). The changes of MEK/ERK in colon tissue and lung tissue showed synchronization variation, or synchronous increased or reduced simultaneously, or increase in lung but decrease in intestinal, or decrease in lung but increase in intestinal.3. The content of TNF-alpha receptor, IL-1beta receptor and C-FOS:In pulmonary disease (CB) model rats, the content of TNF-alpha receptor and IL-1beta receptor in lung tissue were significantly reduced at model70th, but significantly increased in colon tissue at model70th (P<0.05or P<0.01). The content of C-Fos was significantly reduced both in lung and colon tissue at model50th (P<0.05).In bowel disease (UC) model rats, the content of TNF-alpha receptor was decreased in colon tissue but increased in lung tissue significantly at model8th (P＜0.05). At model8th and29th, the content of IL-1beta receptor was increased in colon tissue but decreased in lung tissue significantly (P＜0.05). The content of C-Fos was increased in colon tissue but decreased in lung tissue significantly at model29th and50th (P＜0.01).Conclusion:1. Pulmonary disease (CB) model rats’intestinal flora was changed. There showed disorders of micro-ecological system in respiratory and intestinal system when lung disease spread to the colon. There is a certain degree of dynamic correlation of respiratory and intestinal micro-ecological system. The micro-ecological of the lung and large intestine interaction may be one of the connotations of "the lung and the large intestine being interior-exteriorly related"2. Bowel disease (UC) model rats’respiratory tract flora was changed. When the bowel disease spread to the lung, part of respiratory and intestinal flora synchronization regular changed in bowel disease model rats, or synchronous increased, or reduced simultaneously. This showed that the microcosm’s flora changes may be one of the mechanisms and manifestations of "bowel disease spread to the lung" 3. Pulmonary disease rats have certain regular changes in the MEK/ERK pathway in the lung tissues. P-ERK1/2may be indicators of chronic bronchitis obvious changes in the MEK/ERK pathway. This prompts that MEK/ERK pathway may be one of the biological basis of "lung disease spread to the colon".4. Bowel disease rats showed similar change trends of MEK/p-MEK and ERK/p-ERK in the lung and colon tissue. To some extent reflects the synchronization between the changes in the lung and intestine. This prompts that MEK/ERK pathway may be one of the biological basis of "bowel disease spread to the lung".5. pulmonary disease rats’TNF-alpha receptor, IL-1beta receptor and c-Fos content appears synchronous dynamic change in the lung and intestine tissue, or decreased both in the lung and intestinal simultaneously, or decreased in the lung while increased in the intestine. This prompts that the contents of TNF-alpha receptor, IL-lbeta receptor and c-Fos may be one of the material bases of "lung disease spread to the colon".6. Bowel disease rats’TNF-alpha receptor, IL-1beta receptor and c-Fos were synchronous dynamic changed in the lung and intestine, or decreased both in the lung and intestinal simultaneously, or decreased in intestinal but increased in the lung, or increased in the intestine but decreased in the lung. This prompts that the contents of TNF-alpha receptor, IL-lbeta receptor and c-Fos may be one of the material bases of "bowel disease spread to the lung".更多还原