两种微生态学动物模型的建立及其在抗菌药物残留安全评估中的应用研究

发布时间：2018-06-29 06:03

本文选题：HFA小鼠模型 + 幼年小鼠模型　；参考：《第三军医大学》2009年博士论文

【摘要】： 研究发现人体长期摄入残留剂量抗菌药物会对肠道菌群产生不利影响,如破坏肠道菌群定植抗力,诱导及促进耐药菌的增长等。国际组织、欧美等国家已将抗菌药物残留对人体肠道菌群的影响列为安全性评价的一项重要内容。我国动物性食品抗菌药物残留严重,在这种紧迫形势下,国内已经积极开展相关工作。人肠道菌群定植小鼠模型模拟了人肠道菌群的特性,是抗菌药物残留安全性评价中推荐使用的动物模型。然而,该模型以稳定的成年人肠道菌群为研究对象,致使其评价结果不能反映抗菌药物对脆弱的婴幼儿肠道菌群的影响。因此,有必要建立敏感动物模型用以评估抗菌药物对幼年期肠道菌群的影响,幼年小鼠模型则可满足这一需求。为适应我国抗菌药物残留微生物安全评估工作顺利开展的需要,本课题拟首先依据动物模型构建的相似性原则,建立人肠道菌群定植小鼠模型,并利用该模型评估恩诺沙星残留对人体肠道菌群的影响;其次,依据动物模型构建的敏感性原则,利用幼年小鼠模型研究阿莫西林对幼年期肠道菌群正常发育的长期影响。以上结果将为我国建立更为科学、全面的抗菌药物残留安全性评估体系提供良好动物模型、先进检测技术和基础数据。课题分为二部分: 第一部分目的:建立HFA-KM小鼠模型,研究该模型肠道菌群与人肠道菌群的相似性,为抗菌药物残留微生物学安全评价提供良好的动物模型;评估恩诺沙星残留对HFA-KM小鼠模型肠道菌群数量、组成多样性和耐药性的影响,为我国抗菌药物残留微生物学安全评估工作提供可借鉴的评估模型和技术。方法:(1)对临产KM孕鼠进行无菌剖腹产手术,在无菌隔离器内对剖出的无菌仔鼠进行人工喂养,从而培育无菌KM小鼠。(2)将健康志愿者粪便悬液菌群灌胃接种于无菌KM小鼠肠道,获得HFA-KM小鼠模型。(3)利用PCR-DGGE技术分析HFA-KM小鼠肠道菌群总菌及优势种属—拟杆菌属的菌群组成与志愿者个体肠道菌群的相似性。(4)通过随机饮水方式,连续35d分别给予HFA-KM小鼠高、中、低剂量的恩诺沙星,模拟残留剂量抗菌药物的持续作用,低剂量对应于恩诺沙星的ADI值。(5)活菌计数方法研究HFA-KM小鼠肠道菌群数量和耐药性的变化、PCR-DGGE技术研究肠道菌群结构多样性变化。结果:(1)培育出的无菌KM小鼠经第三方一中国药品生物制品检定所进行无菌检验,检验结果为阴性,证明我们培育的小鼠是无菌小鼠,为HFA小鼠模型的建立提供了动物来源。(2)V3-DGGE图谱及拟杆菌属DGGE图谱显示:尽管两株毛螺旋菌科(Lachnospiraceae)细菌和一株普通拟杆菌(Bacteriodes vulgatus)不能在HFA小鼠肠道中优势定植,但健康志愿者肠道菌群的优势菌大部分可在HFA小鼠肠道中稳定定植,说明本实验部分建立的HFA小鼠模型的肠道菌群具备了健康志愿者肠道菌群的主要特征。(3)中、高剂量的恩诺沙星在给药后期能减少HFA-KM小鼠的总需氧菌和肠杆菌数量,与对照组相比差异显著(P0.05),对其他类群的细菌无显著作用;低剂量的恩诺沙星对肠道菌群的数量无显著影响。(4)中、高剂量的恩诺沙星在给药后期能增加HFA-KM小鼠的总需氧菌和总厌氧菌的耐药性,与对照组相比差异显著(P0.05),对其他类群的细菌无显著作用;低剂量的恩诺沙星对肠道菌群的耐药性无显著影响。(5)V3-DGGE图谱显示,三个剂量的恩诺沙星均显著减小HFA-KM小鼠肠道菌群的丰富度和Shannon指数(P0.05)。第二部分目的:利用幼年小鼠模型研究阿莫西林对肠道菌群正常发育的长期影响,尤其是观察对幼年期肠道优势菌群—乳杆菌和双歧杆菌多样性的影响,为探索幼年小鼠模型在抗菌药物残留安全性评价中的适用性提供动物实验数据。方法:(1)7日龄幼年BALB/C仔鼠连续灌胃14天治疗剂量阿莫西林或生理盐水,于21日龄(灌胃结束时)和56日龄(结束后5周)分别处死5只小鼠,无菌采集小鼠结肠内容物。(2)建立BALB/C小鼠肠道乳杆菌群和双歧杆菌菌群16S rRNA基因克隆文库。(3)PCR-DGGE技术分析肠道总菌、乳杆菌及双歧杆菌菌群结构多样性变化。(4)实时定量PCR技术分析乳杆菌属及双歧杆菌属数量变化。结果:(1)BALB/C小鼠肠道乳杆菌菌群和双歧杆菌菌群结构简单,肠道乳杆菌菌群的优势菌为L. murinus (61.2%)和L. johnsonii (29.5%),双歧杆菌菌群的优势菌为B. pseudolongum (88.5%)。(2)阿莫西林作用于幼年小鼠肠道菌群,停药5周后,小鼠肠道菌群丰富度和Shannon指数仍显著低于正常对照(P0.05)。具体来说,肠道乳杆菌属数量恢复至正常水平,但菌群丰富度和Shannon指数仍显著低于正常对照(P0.05),典型特征为一株优势菌L. johnsonii不能继续定植于小鼠肠道;双歧杆菌属数量显著低于正常对照(P0.05),但对菌群微生物多样性无显著影响。主要结论: 1.将健康志愿者粪便悬液菌群接种于无菌KM小鼠后获得HFA-KM小鼠模型。健康志愿者和HFA-KM小鼠的肠道菌群相似性分析结果表明健康志愿者肠道菌群的优势菌大部分可在HFA-KM小鼠肠道中定植,HFA-KM小鼠模型具备了健康志愿者肠道菌群的主要特征,是抗菌药物残留微生物学安全性评价的良好动物模型。 2.对应于现行恩诺沙星ADI值的药物剂量对肠道菌群数量和耐药性无显著影响,但显著减少了菌群多样性。从肠道菌群多样性角度考虑,现行恩诺沙星ADI值的安全性有待进一步检验。 3.阿莫西林对幼年小鼠肠道菌群的正常发育具有长期干扰作用,尤其是乳杆菌属和双歧杆菌属。说明幼年小鼠模型能敏感的反映出抗菌药物对幼年期肠道菌群的影响,是现有抗菌药物残留微生物学安全性评价动物模型的有益补充。
[Abstract]:It is found that the long - term anti - microbial drugs ingested by the human body can adversely affect the intestinal flora , such as destroying the colonization resistance of the intestinal flora , inducing and promoting the growth of the resistant bacteria , etc . The international organizations , the United States and the like have listed the effects of the antibiotic residues on the intestinal flora of the human body as an important part of the safety evaluation .

However , it is necessary to establish a sensitive animal model to evaluate the effect of antibacterial drugs on the intestinal flora of young children . Therefore , it is necessary to establish a sensitive animal model to evaluate the effect of antibacterial drugs on the intestinal flora of young children .

In order to meet the needs of the successful evaluation of antimicrobial drug residues in China , this paper intends to establish a mouse model of human intestinal flora according to the similarity principle established by animal model , and use the model to evaluate the long - term effects of Ennofloxacin residue on the intestinal flora of human body .

the first portion

Objective : To establish a model of HFA - KM mice , to study the similarity between the intestinal flora and human intestinal flora in the model , to provide a good animal model for the safety evaluation of antimicrobial drug residues .

Methods : ( 1 ) The sterile KM mice were cultured in sterile KM mice by performing aseptic caesarean section on KM pregnant rats .

Results : ( 1 ) The sterile KM mice were tested for sterility by the third party , and the results were negative . The results showed that the mice which were cultured were sterile mice . The results showed that the intestinal flora of the healthy volunteers could decrease the total aerobic bacteria and the number of intestinal bacteria in the healthy volunteers .

the second part

Objective : To study the long - term effects of amoxicillin on the normal development of intestinal flora in young mouse model , in particular to observe the effect of amoxicillin on the diversity of intestinal microflora , Lactobacillus and Bifidobacterium , and to provide animal experiment data for the purpose of exploring the applicability of juvenile mouse model in the evaluation of antimicrobial drug residue safety .

Methods : ( 1 ) BALB / C mice were treated with amoxicillin or normal saline at the age of 21 days ( at the end of the gavage ) and 56 days old ( 5 weeks after the end of the gavage ) .

Results : ( 1 ) The intestinal flora of BALB / C mice and Bifidobacterium strains were simple , the dominant bacteria of the lactobacillus group were L . murinus ( 61.2 % ) and L .

Main conclusions :

1 . The animal model of HFA - KM mice was obtained by inoculating the fecal suspension of healthy volunteers into sterile KM mice . The intestinal flora similarity analysis of healthy volunteers and HFA - KM mice showed that most of the dominant bacteria in healthy volunteers were colonised in the intestinal tract of HFA - KM mice , and the HFA - KM mice model had the main characteristics of the intestinal flora of healthy volunteers .

2 . There were no significant effects on the number and drug resistance of the intestinal flora corresponding to the current Ennofloxacin ADI values , but the diversity of the flora was significantly reduced . From the perspective of the diversity of the intestinal flora , the safety of the current Ennofloxacin ADI value was to be further examined .

3 . Amoxylin has long - term effects on the normal development of intestinal flora in young mice , especially the genus Lactobacillus and Bifidobacterium . It is suggested that the mouse model can sensitively reflect the influence of antibacterial drugs on the intestinal flora of young mice , which is a beneficial complement to the animal model of the evaluation of the residual microbiological safety of antibacterial drugs .
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R-332

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