發(fā)布時間：2018-04-05 18:14

  本文選題：腸道菌群　切入點：多樣性　出處：《第三軍醫(yī)大學》2007年博士論文

【摘要】： 抗生素的不當使用將直接危害人體健康:服用治療劑量抗生素會增加臨床病人二重感染的發(fā)病幾率,被動攝入殘留劑量的抗生素則有可能導致腸道菌群失調(diào)癥的發(fā)生。這兩大類疾病在本質(zhì)上都與抗生素導致的腸道菌群變化密切相關,因此,基于微生態(tài)學基本觀點開展抗生素對腸道菌群作用的深入研究,對于預防和治療相關疾病具有重要意義。 能夠充分模擬抗生素與腸道菌群作用的標準化動物模型是闡述相關疾病發(fā)生機理和探索防治策略的重要基礎,而菌株選擇是微生態(tài)療法發(fā)揮有效性的關鍵。因此,本研究建立了兩大類標準化動物模型以充分模擬人暴露于抗生素的兩種主要途徑,使用體外篩選獲得的乳桿菌菌株對抗生素誘導的菌群失衡進行調(diào)節(jié),結(jié)合經(jīng)典技術與現(xiàn)代分子微生態(tài)技術評價乳桿菌調(diào)節(jié)菌群的有效性,以期闡明腸道微生態(tài)區(qū)系改變與感染發(fā)生的關系,同時為防治菌群失調(diào)和感染提供實驗數(shù)據(jù)。課題分為三部分: 第一部分 目的:構(gòu)建不同劑量頭孢曲松誘導的小鼠腸道菌群失衡模型,分別為研究微生態(tài)區(qū)系與感染之間的關系和殘留劑量抗生素對菌群的影響提供標準化的實驗工具和整體評價體系。 方法:(1)通過灌胃方式,連續(xù)5d給予SPF小鼠頭孢曲松,人為灌胃白假絲酵母菌造成小鼠腸道真菌過增殖模型;(2)通過隨機飲水方式,連續(xù)45d分別給予SPF小鼠三個濃度的低劑量頭孢曲松,模擬殘留劑量抗生素的持續(xù)作用;(3)革蘭染色光鏡和掃描電境直接觀察小鼠菌群變化、活菌計數(shù)研究菌群數(shù)量變化及對白假絲酵母的清除作用、PCR-DGGE技術研究菌群結(jié)構(gòu)多樣性變化、模擬腸道離體模型研究菌群定植抗力的變化。 結(jié)果:(1)50mg/d的頭孢曲松會導致小鼠腸道菌群重度紊亂,厭氧總菌、乳桿菌和腸球菌在處理期間數(shù)量出現(xiàn)極顯著下降(P0.01),雙歧桿菌和腸桿菌缺如(P0.001);DGGE圖譜顯示,總菌和乳桿菌豐富度和多樣性極顯著低于正常水平(P0.01),腸球菌多樣性指數(shù)也出現(xiàn)顯著下降(P0.05);通過對DGGE條帶進行回收、測序,發(fā)現(xiàn)Bacillus sp.部分菌株失去優(yōu)勢菌群的地位,一株Lactobacillus reuteri演替為優(yōu)勢菌;腸黏膜表面膜菌群脫落,腸道菌群定植抗力嚴重受損。(2)300μg/ml及30μg/ml CRO水溶液持續(xù)處理45d會導致小鼠腸道菌群紊亂,厭氧總菌、乳桿菌和腸桿菌數(shù)量均出現(xiàn)先降后升的趨勢,而腸桿菌數(shù)量異常增殖,雙歧桿菌數(shù)量顯著減少(P0.01)且無法恢復,腸球菌無顯著變化(P0.05);3μg/ml頭孢曲松處理對小鼠腸道菌群數(shù)量無顯著影響(P0.05);DGGE分析可見總菌和乳桿菌菌群多樣性顯著下降,腸球菌多樣性指數(shù)無顯著變化;通過對DGGE條帶進行回收、測序,發(fā)現(xiàn)處理中期出現(xiàn)兩株Ruminococcus torques,但其數(shù)量未能增殖,而對頭孢曲松耐藥的一株Enterococcus faecium優(yōu)勢效應貫穿處理始終;小鼠腸道乳桿菌的優(yōu)勢菌群為兩株Lactobacillus reuteri和兩株Lactobacillus salivarius subsp. Salivarius,數(shù)量優(yōu)勢不受頭孢曲松作用的影響;定植抗力受損,但仍可顯著抑制白假絲酵母的增殖(P0.05)。 第二部分 目的:通過體外模型篩選出拮抗白假絲酵母能力最強的乳桿菌用于體內(nèi)實驗,研究乳桿菌對小鼠腸道失衡菌群的調(diào)節(jié)效果。 方法:(1)結(jié)晶紫芽管染色法比較8株乳桿菌及其代謝產(chǎn)物體外拮抗白假絲酵母芽管形成的能力,共孵育法和瓊脂打孔擴散法比較8株乳桿菌活菌與培養(yǎng)乏液對白假絲酵母的生長抑制能力;(2)對治療劑量抗生素作用后的小鼠,于不同時間點,采用預防、同步、治療三種策略,通過灌胃方式給予腸道白假絲酵母過增殖小鼠模型108CFU的LGG;對低劑量抗生素作用的小鼠,向飲用頭孢曲松水溶液中直接混合109CFU乳桿菌LGG;(3)革蘭染色光鏡直接觀察小鼠菌群變化、活菌計數(shù)研究菌群數(shù)量變化及對白假絲酵母的清除作用、PCR-DGGE技術研究菌群結(jié)構(gòu)多樣性變化、模擬腸道離體模型研究菌群定植抗力的變化。 結(jié)果:(1)鼠李糖乳桿菌LGG對白假絲酵母的芽管抑制及生長抑制能力均顯著高于其他乳桿菌;短鏈脂肪酸中只有丁酸能夠顯著抑制白假絲酵母芽管的形成;(2)LGG對小鼠重度失衡的菌群調(diào)節(jié)作用明顯,可顯著促進厭氧總菌、雙歧桿菌和乳桿菌數(shù)量的恢復;LGG干預后,受損菌群的豐富度和乳桿菌的多樣性指數(shù)恢復至正常(P0.05),且預防處理恢復菌群的效果最顯著;定植抗力顯著恢復,LGG干預后的小鼠可以極顯著清除腸道內(nèi)的白假絲酵母,仍以預防處理組效果最好(3)LGG調(diào)節(jié)殘留劑量頭孢曲松誘導的菌群失調(diào)具有一定效果,促進了雙歧桿菌數(shù)量的恢復,但無法抑制腸桿菌的過度增殖;可以恢復乳桿菌的豐富度和多樣性,但總菌多樣性指數(shù)無顯著變化(P0.05),對腸球菌的數(shù)量和多樣性均無顯著影響(P0.05);能夠顯著增強菌群定植抗力,LGG干預組對于白假絲酵母的清除能力極顯著的高于抗生素單獨處理組(P0.01)。 第三部分 目的:對暴露在抗生素壓力下的乳桿菌介導耐藥性傳遞的風險進行評估。 方法:(1)K-B法檢測8株乳桿菌對于14種抗生素的敏感性;(2)雙紙片擴散法檢測體內(nèi)實驗菌株LGG產(chǎn)超廣譜β-內(nèi)酰胺酶的情況;(3)提取體內(nèi)實驗菌株LGG的質(zhì)粒,進行菌體內(nèi)可傳遞耐藥因子的檢測。 結(jié)果:(1)8株乳桿菌對抗生素表現(xiàn)出多重耐藥,LGG可以耐受5種受試藥物,而耐受萬古霉素的乳桿菌菌株為7株;(2)LGG不產(chǎn)超廣譜β-內(nèi)酰胺酶,不含質(zhì)粒。 主要結(jié)論: 1、臨床劑量頭孢曲松的作用可嚴重破壞小鼠腸道菌群的平衡,該模型可以成功模擬臨床病人服用頭孢曲松進而造成腸道真菌過增殖的現(xiàn)象。頭孢曲松作用后,小鼠腸道菌數(shù)量顯著減少、多樣性降低、菌群固有定植抗力嚴重受損,白假絲酵母菌可大量增殖。因此,大劑量頭孢曲松誘導的小鼠腸道菌群失衡模型證明了腸道菌群紊亂與腸源性感染具備因果關系。 2、食品殘留劑量頭孢曲松的長期作用可導致小鼠腸道菌群出現(xiàn)輕度失衡,該模型成功模擬了人體經(jīng)由食物鏈被動攝入抗生素造成的菌群失調(diào)。 3、通過體外篩選獲得的乳桿菌菌株LGG對兩類模型進行干預,可有效調(diào)節(jié)頭孢曲松導致的腸道菌群失衡,恢復菌群數(shù)量、多樣性和定植抗力;使用乳桿菌調(diào)節(jié)腸道菌群,同時存在耐藥性轉(zhuǎn)移的風險,但乳桿菌LGG株在體內(nèi)介導耐藥性傳遞的風險性較小。 創(chuàng)新性: 1、以腸道微生態(tài)學為著眼點,全面模擬人體暴露于抗生素的兩種不同途徑,首次使用同種抗生素的不同劑量構(gòu)建了兩類腸道菌群失衡模型,并且分別研究了乳桿菌對于不同程度菌群失衡的調(diào)節(jié)作用,具有微生態(tài)學特色的整體、系統(tǒng)化實驗設計具有一定創(chuàng)新性; 2、使用臨床劑量的頭孢曲松處理小鼠,成功建立了腸道白假絲酵母局部感染的小鼠模型,以微生態(tài)學觀點闡述了抗生素誘導的腸道菌群失衡與感染發(fā)生發(fā)展的關系; 3、利用乳桿菌LGG株穩(wěn)定和恢復腸道菌群的方法來防治感染,為抗感染治療提供了新的參考策略,為微生態(tài)療法運用于抗感染治療提供了理論基礎,同時為本菌株開發(fā)為微生態(tài)制劑提供了新的實驗依據(jù)。 4、結(jié)合傳統(tǒng)活菌計數(shù)與現(xiàn)代分子微生態(tài)學PCR-DGGE技術,為研究抗生素對于腸道菌群的影響及其防治提供了新的評價方法; 5、本實驗在研究乳桿菌調(diào)節(jié)菌群有效性的同時,對其介導腸道菌群之間耐藥性傳遞的風險進行了初步評估,對乳桿菌耐藥性的關注具有前瞻性。
[Abstract]:The improper use of antibiotics will directly harm human health : the administration of therapeutic doses of antibiotics will increase the incidence of secondary infection in clinical patients , and the passive intake of residual doses of antibiotics may lead to the occurrence of intestinal dysbacteriosis . These two major diseases are intrinsically related to the changes in intestinal flora caused by antibiotics . Therefore , it is important to study the effect of antibiotics on intestinal flora based on the basic viewpoint of microecology .






A standardized animal model which can fully simulate the effects of antibiotics and intestinal flora is an important basis for the pathogenesis of related diseases and the exploration and prevention strategies , and the selection of strains is the key to the effectiveness of micro - ecological therapy . Therefore , this study establishes two major types of standardized animal models to fully simulate the two main routes of human exposure to antibiotics .






the first portion






Objective : To establish a model of intestinal flora imbalance induced by different doses of cephalosporin , and to provide a standardized experimental tool and an overall evaluation system for the study of the relationship between microecological region and infection and the influence of residual dose antibiotics on the flora .






Methods : ( 1 ) SPF mice were given SPF mice by gavage for 5 days .






Results : ( 1 ) There was a significant decrease in the number of intestinal flora in mice ( P0.01 ) . Salivarius , the superiority of quantity was not influenced by the action of cephalosporin ; the colonization resistance was damaged , but the proliferation of Candida albicans could be significantly inhibited ( P0.05 ) .






the second part






Objective : To study the effect of lactobacillus on the intestinal imbalance flora of mice .






Methods : ( 1 ) The growth inhibition ability of 8 strains of Lactobacillus reuteri and its metabolites in vitro was compared with 8 strains of lactobacillus and its metabolites .






Results : ( 1 ) The inhibition and growth inhibition ability of Lactobacillus rhamnosus LGG was significantly higher than that of other lactobacillus .






PART III






Objective : To evaluate the risk of drug resistance transfer mediated by Lacan exposed to antibiotic pressure .






Methods : ( 1 ) The sensitivity of 8 strains of Lactobacillus to 14 antibiotics was detected by K - B method . ( 2 ) In vivo experiment strain LGG was detected by double - paper diffusion method .






Results : ( 1 ) Eight strains of lactobacillus showed multiple resistance to antibiotics , and LGG could tolerate 5 kinds of tested drugs , and the strains resistant to vancomycin were 7 strains ; ( 2 ) LGG did not produce the super - broad - spectrum 尾 - lactamase and did not contain plasmids .






Main conclusions :






1 . The effect of the clinical dose of Cefomycin can seriously destroy the balance of intestinal flora in mice . The model can successfully simulate the phenomenon of excessive intestinal flora in clinical patients . After the action of cephalosporin , the number of intestinal bacteria in mice is obviously reduced , the diversity is reduced , the inherent colonization resistance of the flora is severely damaged , and the Candida albicans can proliferate . Therefore , the model of intestinal flora imbalance induced by the large - dose cephalosporin has proved the causal relationship between intestinal flora disturbance and enterogenous infection .






2 . The long - term effects of the food residual dose of cephalosporin could lead to mild imbalance in the intestinal flora of mice . The model successfully simulated the dysbacteriosis caused by the passive ingestion of antibiotics through the food chain .






3 . The lactobacillus strain LGG obtained by in vitro screening can effectively regulate the imbalance of intestinal flora , restore the quantity , the diversity and the resistance of the intestinal flora caused by the cephalosporin , and use lactobacillus to adjust the intestinal flora , and meanwhile , the risk of drug resistance transfer exists , but the risk of the LGG strain mediated drug resistance transmission in vivo is small .






Innovative :






1 . Based on the microecology of intestinal tract , two different routes of human exposure to antibiotics were fully simulated . Two kinds of intestinal flora imbalance models were constructed for the first time using different doses of the same antibiotics .






2 . A mouse model of local infection of Candida albicans was successfully established by using the clinical dose of cefsuspine , and the relationship between the imbalance of intestinal flora and the development of infection was described by microecology .






3 . The method for stabilizing and recovering the intestinal flora by using the lactobacillus LGG strain provides a new reference strategy for the anti - infection treatment , and provides a theoretical basis for the application of the micro - ecological therapy to the anti - infection treatment , and provides a new experimental basis for the development of the strain as a micro - ecological preparation .






4 . Combining the traditional living bacteria counting and modern molecular microecology PCR - DGGE technology , this paper provides a new evaluation method for the study of the effect of antibiotics on intestinal flora and the prevention and control of antibiotics .






5 . In this experiment , the risk of drug - resistance transfer between intestinal flora and intestinal flora was preliminarily assessed at the same time of studying the efficacy of Lactobacillus plantarum , which was forward - looking .

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R363;R96

【引證文獻】

相關期刊論文 前2條

1 宋克玉;江振友;嚴群超;陳琛;施珊珊;;黨參及茯苓對小鼠腸道菌群調(diào)節(jié)作用的實驗研究[J];中國臨床藥理學雜志;2011年02期

2 吳青龍;陳廷濤;熊順強;姜淑英;李勝杰;魏華;;利用PCR-DGGE技術監(jiān)測Lactobacillus plantarum WCFS1對小鼠腸道菌群的動態(tài)變化[J];南昌大學學報(理科版);2011年06期

相關碩士學位論文 前4條

1 鐘轉(zhuǎn)華;肝硬化患者腸生物機械屏障改變及微生態(tài)制劑干預價值[D];暨南大學;2010年

2 周敏;乳酸桿菌對重型顱腦損傷大鼠胃腸動力障礙的影響[D];第三軍醫(yī)大學;2010年

3 王尤麗;低聚半乳糖調(diào)節(jié)小鼠腸道菌群的作用[D];鄭州大學;2012年

4 王曉翠;乳酸芽孢桿菌的分離、鑒定及菌制劑制備的研究[D];東北農(nóng)業(yè)大學;2012年

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