發(fā)布時間：2018-06-26 19:46

  本文選題：生命早期應(yīng)激 + 新生期母嬰分離�。� 參考：《東南大學(xué)》2017年碩士論文

【摘要】：[目的]本實(shí)驗(yàn)通過建立生命早期應(yīng)激模型(新生期母嬰分離,NMS)及哮喘雙模型,探討新生期母嬰分離對成年后哮喘氣道炎癥的影響及其機(jī)制,為臨床更好理解生命早期應(yīng)激事件與哮喘的關(guān)系奠定實(shí)驗(yàn)基礎(chǔ);為提倡母嬰同室、家庭整體護(hù)理治療手段提供可靠依據(jù);也為后期預(yù)防應(yīng)激加重哮喘的治療提供新的策略。[方法]建立新生期母嬰分離模型,以Balb/c新生雄鼠為研究對象,隨機(jī)分成NMS組及非NMS組。仔鼠出生當(dāng)天作為PNDO,NMS組在PND2～PND14,每天同時間段分離新生鼠及母鼠3h,而非NMS組不做分離處理。分別于PND21、PND28、PND35、PND42(即斷奶當(dāng)天、一周、兩周、三周)收集糞便,應(yīng)用變性梯度凝膠電泳(DGGE)技術(shù)檢測腸道菌群圖譜,并采用高效液相色譜法獲取腸道菌群代謝產(chǎn)物短鏈脂肪酸(SCFAs)的含量。在PND43采用雞卵清蛋白(OVA)致敏法建立哮喘模型。將非NMS組隨機(jī)分為正常對照組(A組)、哮喘組(B組),NMS組建立哮喘模型成為母嬰分離哮喘組(C組)和短鏈脂肪酸喂養(yǎng)母嬰分離哮喘組(D組)。收集支氣管肺泡灌洗液(BALF)計(jì)數(shù)各組炎癥細(xì)胞總數(shù)及嗜酸性粒細(xì)胞(EOS)數(shù)目;HE染色觀察雄鼠肺組織病理變化;同時用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測雄鼠肺組織中細(xì)胞因子(IL-13、IL-25、IL-33)水平;蛋白質(zhì)免疫印跡法(Western blot)測定肺組織中Occludin和E-cadherin蛋白的表達(dá)。[結(jié)果]1.新生鼠母嬰分離后腸道菌群及SCFAs含量的變化:發(fā)現(xiàn)斷奶后一周、兩周、三周NMS組雄鼠腸道菌群DGGE圖譜的條帶數(shù)S'、多樣性指數(shù)H'和優(yōu)勢度指數(shù)D'均低于非NMS組(P0.05),而斷奶當(dāng)天兩組條帶數(shù)S\、多樣性指數(shù)H'和優(yōu)勢度指數(shù)D'無顯著統(tǒng)計(jì)學(xué)差異。高效液相色譜檢測發(fā)現(xiàn):NMS組和非NMS組間腸道中乙酸、丙酸含量不同,但無統(tǒng)計(jì)學(xué)意義;而非NMS組雄鼠腸道中丁酸的含量明顯高于NMS組,且差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。2.新生鼠母嬰分離后哮喘氣道炎癥的變化:C組雄鼠BALF中炎癥細(xì)胞總數(shù)及EOS數(shù)目高于B組及A組,D組較C組有所減少(P0.05)。C組雄鼠肺組織HE染色可見肺間質(zhì)內(nèi)有較多以EOS為主的炎細(xì)胞浸潤;而B組及D組上述病理改變減輕,A組未見明顯變化。B組肺組織中細(xì)胞因子(IL-13、IL-25、IL-33)水平均明顯高于A組但低于C組,而C組高于D組,且差異均具有統(tǒng)計(jì)學(xué)意義。3.新生鼠母嬰分離引起哮喘氣道炎癥變化可能存在的機(jī)制:C組雄鼠肺組織HE染色可見支氣管上皮杯狀細(xì)胞輕度增多,管腔內(nèi)有少量黏性分泌物;而B組及D組上述病理改變減輕,A組未見明顯改變。Western blot顯示:B、C、D三組肺組織Occludin和E-cadherin蛋白的表達(dá)較A組明顯下降,其中C組蛋白表達(dá)水平低于B組,而D組高于C組,且差異具有統(tǒng)計(jì)學(xué)意義。[結(jié)論]新生期母嬰分離可影響子代腸道菌群的多樣性并能降低腸道中丁酸的含量。新生期母嬰分離的雄鼠也可出現(xiàn)成年后哮喘氣道炎癥加重的現(xiàn)象,引起這一現(xiàn)象的機(jī)制可能是:生命早期腸道菌群多樣性的改變引起了短鏈脂肪酸含量的減少,而短鏈脂肪酸含量的減少進(jìn)一步破壞了氣道上皮屏障,最終加重哮喘。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R562.25

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