本文關(guān)鍵詞： 新生兒敗血癥和腦膜炎 大腸桿菌K1株 腸道屏障 血腦屏障 益生菌代謝產(chǎn)物 黏蛋白 免疫球蛋白A　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：一、研究背景與目的敗血癥和腦膜炎是導(dǎo)致新生兒,特別是早產(chǎn)兒死亡的重要原因。大腸桿菌(Escherichia coli,E.coli)是引起新生兒敗血癥和腦膜炎最常見(jiàn)的革蘭陰性致病菌。新生兒E.coli敗血癥和腦膜炎的治療主要依靠抗生素,但療效方面有著諸多的限制,其不合理的使用也導(dǎo)致耐藥E.coli感染發(fā)生率逐年上升。因此,亟需積極的開(kāi)發(fā)預(yù)防新生兒E.coli敗血癥和腦膜炎的新方法,代替以抗生素為主的治療方法。大腸桿菌是腸道常駐菌,成功突破腸黏膜屏障是自然狀態(tài)下E.coli引起敗血癥和腦膜炎的關(guān)鍵步驟。新生兒的腸道屏障功能發(fā)育不完全,極易感染E.coli繼而引發(fā)敗血癥和腦膜炎,因此,通過(guò)外界干預(yù)增強(qiáng)新生兒腸道屏障功能是預(yù)防該病的有效措施。目前的研究表明,鼠李糖乳桿菌GG株(Lactobacillus rhamnosusGG,LGG)的代謝產(chǎn)物具有調(diào)節(jié)腸道屏障功能的效果,但能否用于預(yù)防新生兒E.coli感染仍未有研究。本研究著重探討了 LGG培養(yǎng)上清液(LGG Culture Supernatant,LCS)預(yù)防新生兒E.coli感染的效果及相關(guān)機(jī)理,并對(duì)代謝產(chǎn)物中的有效活性成分作了初步分析。二、研究方法和結(jié)果方法:首先,培養(yǎng)人結(jié)腸腺癌細(xì)胞Caco-2建立體外腸屏障模型,初步研究LCS保護(hù)腸屏障功能并預(yù)防E.coli易位的效果;其次,建立新生大鼠腸源性E.coli感染模型,進(jìn)一步研究LCS預(yù)防新生大鼠E.coli系統(tǒng)性感染的效果和相關(guān)機(jī)理;最后,通過(guò)高效液相色譜-串聯(lián)質(zhì)譜等技術(shù),分析并初步鑒定LCS中發(fā)揮功效的活性成分。結(jié)果:體外研究結(jié)果顯示LCS能抑制E.coli降解Caco-2分泌的黏蛋白,抑制E.coli的黏附和侵襲,減輕E.coli對(duì)腸上皮細(xì)胞間緊密連接的破壞并阻止其易位;體內(nèi)研究結(jié)果顯示LCS能有效預(yù)防新生大鼠腸源性E.coli感染,其機(jī)制是促進(jìn)了新生大鼠腸道細(xì)胞的增殖和分化、增加了腸道表層的黏蛋白、免疫球蛋白A和細(xì)胞間緊密連接蛋白的表達(dá)并降低了腸道的透通性。這些研究結(jié)果表明LCS能促進(jìn)新生大鼠腸道屏障功能的形成和完善,并藉此增強(qiáng)大鼠對(duì)腸源性E.coli感染的抵抗力。最后,通過(guò)高效液相色譜-串聯(lián)質(zhì)譜和蛋白表達(dá)與純化技術(shù),我們鑒定并體外表達(dá)和純化了 LCS中的一種假想蛋白HMPREF0539_2242,后續(xù)的研究發(fā)現(xiàn)該蛋白很可能發(fā)揮了促進(jìn)新生大鼠腸道屏障功能成熟的作用。三、結(jié)論新生兒早期腸道屏障功能的不完善主要表現(xiàn)為腸道機(jī)械屏障未形成、正常菌群結(jié)構(gòu)未建立和腸道免疫系統(tǒng)不成熟,這些都為E.coli感染提供了可乘之機(jī)。在本研究中,我們首次發(fā)現(xiàn)LGG的代謝產(chǎn)物可促進(jìn)新生大鼠腸道機(jī)械屏障和免疫屏障的形成,繼而增強(qiáng)大鼠對(duì)E.coli感染的抵抗力。通過(guò)質(zhì)譜分析,我們鑒定并純化了 LCS中的一種蛋白,發(fā)現(xiàn)該蛋白很可能發(fā)揮了上述的益生作用。這些研究結(jié)果為開(kāi)發(fā)新生兒E.coli敗血癥和腦膜炎的新型預(yù)防藥物提供了基礎(chǔ)。
[Abstract]:First, the background and purpose of the study was that septicemia and meningitis were the cause of newborns, E. coli Escherichia coli is the most common gram-negative pathogen causing neonatal septicemia and meningitis. The treatment of neonatal E. coli septicemia and meningitis mainly depends on antibiotics. However, there are many limitations in therapeutic effect, and its irrational use also leads to the increasing incidence of drug-resistant E. coli infection year by year. Therefore, it is urgent to actively develop new methods to prevent neonatal E. coli septicemia and meningitis. Escherichia coli is a resident enteric bacteria, and it is a key step for E. coli to successfully break through intestinal mucosal barrier in natural state to cause septicemia and meningitis. The intestinal barrier function of newborn is not fully developed. E. coli is highly susceptible to septicemia and meningitis. Therefore, enhancing the intestinal barrier function of newborns through external intervention is an effective measure to prevent the disease. The metabolites of Lactobacillus rhamnosus GGGGG have the function of regulating intestinal barrier. However, whether it can be used to prevent neonatal E. coli infection has not been studied. In this study, the effect of LGG culture supernatant, LGG Culture supernatant LCSC, on the prevention of neonatal E. coli infection and its related mechanism were discussed, and the effective active components in metabolites were preliminarily analyzed. Methods and results: firstly, the intestinal barrier model was established by cultured human colon adenocarcinoma cell line Caco-2, and the effect of LCS on protecting intestinal barrier function and preventing E. coli translocation was preliminarily studied. To further study the efficacy and related mechanism of LCS in preventing systemic infection of E. coli in newborn rats. Finally, by means of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS), Results: in vitro studies showed that LCS could inhibit the degradation of mucin secreted by Caco-2 and inhibit the adhesion and invasion of E. coli. The results of in vivo study showed that LCS could effectively prevent enterogenic E. coli infection in neonatal rats, and its mechanism was to promote the proliferation and differentiation of intestinal cells in neonatal rats. It increased the expression of mucin, immunoglobulin A and intercellular tight junction protein, and decreased the permeability of intestinal tract. These results suggest that LCS can promote the formation and improvement of intestinal barrier function in neonatal rats. Finally, high performance liquid chromatography-tandem mass spectrometry (HPLC / MS) and protein expression and purification techniques were used to enhance the resistance of rats to enterogenic E. coli infection. We have identified, expressed and purified a hypothetical protein HMPREF05392242 from LCS in vitro. Subsequent studies have found that HMPREF05392242 may play a role in promoting the maturation of intestinal barrier function in neonatal rats. Conclusion the imperfections of intestinal barrier function in the early stage of newborn mainly show that the intestinal mechanical barrier is not formed, the normal flora structure is not established and the intestinal immune system is not mature, which provide the opportunity for E. coli infection. We found for the first time that the metabolites of LGG could promote the formation of intestinal mechanical and immune barrier in neonatal rats, and then enhance the resistance of rats to E. coli infection. By mass spectrometry, we identified and purified a protein in LCS. These results provide the basis for the development of new prophylaxis of neonatal E. coli septicemia and meningitis.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R722.1

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;某些因素對(duì)大鼠胃肌電的影響[J];基礎(chǔ)醫(yī)學(xué)與臨床;2001年S1期

2 陳同度,張昌穎;素食大鼠的貧血現(xiàn)象[J];營(yíng)養(yǎng)學(xué)報(bào);1957年04期

3 陳偉強(qiáng);趙善廣;;自制注射用大鼠固定裝置[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);1992年04期

4 肖柳英,林培英,馮昭明,張丹;不同周齡的SD大鼠生理、生化及體重的正常值測(cè)定[J];中藥新藥與臨床藥理;1996年03期

5 李淑云;簡(jiǎn)易大鼠灌胃器的制作[J];錦州醫(yī)學(xué)院學(xué)報(bào);2001年04期

6 楊明智,陳積圣;一種大鼠抓取與固定的新工具介紹[J];上海實(shí)驗(yàn)動(dòng)物科學(xué);2001年03期

7 戴英,陸群;復(fù)方H_(505)對(duì)Wistar大鼠外周血的血液流變學(xué)指標(biāo)的影響[J];中國(guó)血液流變學(xué)雜志;2001年01期

8 韋應(yīng)波,孫喜慶,曹新生,姚永杰,馮岱雅,楊長(zhǎng)斌;+Gz暴露時(shí)間對(duì)大鼠記憶功能和行為的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年01期

9 呂學(xué)軍,郭俊生,李敏,周利梅,張永娟;暈船大鼠體內(nèi)鐵含量的變化[J];中國(guó)職業(yè)醫(yī)學(xué);2003年04期

10 湯仁仙,王迎偉,王慧,周峰;201A中藥合劑對(duì)大鼠抗腎小球基底膜腎炎病變的影響[J];徐州醫(yī)學(xué)院學(xué)報(bào);2003年06期

相關(guān)會(huì)議論文 前10條

1 尹音;孫振宇;胡敏;李冬霞;;持續(xù)性高正加速度對(duì)大鼠顳頜關(guān)節(jié)損傷的作用[A];第八屆全國(guó)顳下頜關(guān)節(jié)病學(xué)及（牙合）學(xué)大會(huì)論文匯編[C];2011年

2 祝~=驤;iJ梊霞;洃克琴;崔素英;文允摪;，

本文編號(hào)：1553827