新生鼠母婴分离对成年后哮喘气道炎症的影响

发布时间：2018-06-26 19:46

本文选题：生命早期应激 + 新生期母婴分离　；参考：《东南大学》2017年硕士论文

【摘要】：[目的]本实验通过建立生命早期应激模型(新生期母婴分离,NMS)及哮喘双模型,探讨新生期母婴分离对成年后哮喘气道炎症的影响及其机制,为临床更好理解生命早期应激事件与哮喘的关系奠定实验基础;为提倡母婴同室、家庭整体护理治疗手段提供可靠依据;也为后期预防应激加重哮喘的治疗提供新的策略。[方法]建立新生期母婴分离模型,以Balb/c新生雄鼠为研究对象,随机分成NMS组及非NMS组。仔鼠出生当天作为PNDO,NMS组在PND2～PND14,每天同时间段分离新生鼠及母鼠3h,而非NMS组不做分离处理。分别于PND21、PND28、PND35、PND42(即断奶当天、一周、两周、三周)收集粪便,应用变性梯度凝胶电泳(DGGE)技术检测肠道菌群图谱,并采用高效液相色谱法获取肠道菌群代谢产物短链脂肪酸(SCFAs)的含量。在PND43采用鸡卵清蛋白(OVA)致敏法建立哮喘模型。将非NMS组随机分为正常对照组(A组)、哮喘组(B组),NMS组建立哮喘模型成为母婴分离哮喘组(C组)和短链脂肪酸喂养母婴分离哮喘组(D组)。收集支气管肺泡灌洗液(BALF)计数各组炎症细胞总数及嗜酸性粒细胞(EOS)数目;HE染色观察雄鼠肺组织病理变化;同时用酶联免疫吸附试验(ELISA)检测雄鼠肺组织中细胞因子(IL-13、IL-25、IL-33)水平;蛋白质免疫印迹法(Western blot)测定肺组织中Occludin和E-cadherin蛋白的表达。[结果]1.新生鼠母婴分离后肠道菌群及SCFAs含量的变化:发现断奶后一周、两周、三周NMS组雄鼠肠道菌群DGGE图谱的条带数S'、多样性指数H'和优势度指数D'均低于非NMS组(P0.05),而断奶当天两组条带数S\、多样性指数H'和优势度指数D'无显著统计学差异。高效液相色谱检测发现:NMS组和非NMS组间肠道中乙酸、丙酸含量不同,但无统计学意义;而非NMS组雄鼠肠道中丁酸的含量明显高于NMS组,且差异具有统计学意义(P0.01)。2.新生鼠母婴分离后哮喘气道炎症的变化:C组雄鼠BALF中炎症细胞总数及EOS数目高于B组及A组,D组较C组有所减少(P0.05)。C组雄鼠肺组织HE染色可见肺间质内有较多以EOS为主的炎细胞浸润;而B组及D组上述病理改变减轻,A组未见明显变化。B组肺组织中细胞因子(IL-13、IL-25、IL-33)水平均明显高于A组但低于C组,而C组高于D组,且差异均具有统计学意义。3.新生鼠母婴分离引起哮喘气道炎症变化可能存在的机制:C组雄鼠肺组织HE染色可见支气管上皮杯状细胞轻度增多,管腔内有少量黏性分泌物;而B组及D组上述病理改变减轻,A组未见明显改变。Western blot显示:B、C、D三组肺组织Occludin和E-cadherin蛋白的表达较A组明显下降,其中C组蛋白表达水平低于B组,而D组高于C组,且差异具有统计学意义。[结论]新生期母婴分离可影响子代肠道菌群的多样性并能降低肠道中丁酸的含量。新生期母婴分离的雄鼠也可出现成年后哮喘气道炎症加重的现象,引起这一现象的机制可能是:生命早期肠道菌群多样性的改变引起了短链脂肪酸含量的减少,而短链脂肪酸含量的减少进一步破坏了气道上皮屏障,最终加重哮喘。
[Abstract]:[Objective] to establish an early life stress model (neonatal maternal infant separation, NMS) and asthma double model, to explore the effect and mechanism of maternal and infant separation on airway inflammation in adult asthma during the new period, and to lay a foundation for better understanding of the relationship between early life stress events and asthma. The method of treatment provides a reliable basis for the treatment of stress exacerbation of asthma in the later period. [Methods] a new model of neonatal maternal and infant separation was established. The newborn male rats of Balb/c were randomly divided into NMS group and non NMS group. The baby mice were born on the same day as PNDO, NMS group was PND2 to PND14, and the newborn rats and their mothers were separated every day at the same time. Rat 3h, not NMS group did not do separate treatment. The feces were collected from PND21, PND28, PND35, PND42 (one week, two weeks, three weeks), the intestinal flora was detected by denaturing gradient gel electrophoresis (DGGE), and the content of short chain fatty acid (SCFAs) of intestinal microflora was obtained by high performance liquid chromatography. Chicken eggs were used in PND43. The asthma model was established by OVA sensitization method. The non NMS group was randomly divided into the normal control group (group A), the asthma group (group B), the NMS group established the asthma model as the mother to baby separated asthma group (C group) and the short chain fatty acid feeding mother to infant asthma group (D group). The total number of inflammatory cells and the eosinophilic granulocyte count were collected by collecting bronchoalveolar lavage fluid (BALF). The number of EOS, the pathological changes of lung tissue in male rats were observed by HE staining, and the levels of cytokine (IL-13, IL-25, IL-33) in lung tissues of male rats were detected by enzyme linked immunosorbent assay (ELISA), and the expression of Occludin and E-cadherin proteins in lung tissue was determined by protein immunoblotting (Western blot). [results]1. neonatal rats were separated after maternal and infant isolation. And the change of SCFAs content: the number of bands of DGGE Atlas of intestinal microflora in group NMS after weaning, two weeks and three weeks was S', the diversity index H' and dominance index D'were lower than non NMS group (P0.05), but the number of two bands in the day of weaning was S, the diversity index H' and dominance index D'were not statistically significant. High performance liquid chromatography detection found NMS The contents of acetic acid and propionic acid in the intestinal tract between the group and the non NMS group were different, but there was no statistical significance, but the content of butyric acid in the intestinal tract of the non NMS group was significantly higher than that of the NMS group, and the difference was statistically significant (P0.01) the changes of airway inflammation in the neonatal rats after the separation of.2. from the newborn rats: the total number of inflammatory cells and the number of EOS in the BALF of group C male rats were higher than that of the B group and A group, D group Compared with group C, the HE staining of lung tissue in group.C of group.C showed that there were more inflammatory cells in the interstitial lung of the lung, but the pathological changes in B and D groups were reduced, and the level of cytokines in the group of.B (IL-13, IL-25, IL-33) in the group of.B was significantly higher than that in the group of.B (IL-13, IL-25, IL-33), but the group was higher than that in the group, and the difference was all of the group. The possible mechanisms of airway inflammation in.3. neonatal rats were observed. In group C, HE staining in the lung tissue of the male rats showed a slight increase in the goblet cells in the bronchial epithelium and a small amount of mucus secretions in the lumen, while the pathological changes in the B and D groups were relieved, and the A group had no obvious changes in the.Western blot display: B, C, D three groups of lung tissue Occ The expression of Ludin and E-cadherin protein was significantly lower than that in the A group, and the expression level of C group was lower than that of the B group, while the D group was higher than the C group, and the difference was statistically significant. [Conclusion] the separation of the newborn mother and baby can affect the diversity of the intestinal flora and the content of butyric acid in the intestinal tract. The mechanism of exacerbation of airway inflammation in asthma may be the mechanism of this phenomenon: the change in intestinal flora diversity in the early life caused the decrease of short chain fatty acid content, and the decrease of short chain fatty acid content further destroys the airway epithelial barrier and eventually aggravates asthma.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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