【作者】 袁芝琼；

【导师】 申元英；

【作者基本信息】 大理学院 ， 流行病与卫生统计学， 2013， 硕士

【摘要】 目的本课题拟以土霉素及氯霉素标准品，常见化脓性致病菌大肠埃希菌及金黄色葡萄球菌，以及易于获取的常见益生菌乳酸杆菌为研究对象，通过多因素多水平正交设计进行动物实验，在抗生素对小鼠进行菌群失调造模后，以常见的化脓性致病菌对小鼠进行攻击，观察致病菌的作用效果是否有增强。再以益生菌对肠道菌群进行调整，探讨益生菌缓解或恢复肠道菌群失调的效能，寻找影响肠道菌群分布的主要因素，以期为人体肠道微生态健康提供有力的科研证据。方法按照多因素多水平正交设计方案对各实验组分别采用土霉素、氯霉素连续灌胃3d，停药4d后给予不同种类和剂量致病菌连续灌胃7d，停药1d后给予不同剂量乳酸杆菌连续灌胃7d，停药1d后取小鼠盲肠内容物进行菌群选择培养，观察肠道菌群变化。结果（一）正常小鼠肠道菌群分布1.不同窝别小鼠肠道菌群分布实验不同时段中所采用小鼠的乳酸杆菌总数、肠杆菌总数、肠球菌总数及真菌总数差异均无统计学意义（p>0.05）。提示不同窝别的小鼠之间肠道菌群分布无差异，实验有效。2.不同性别小鼠肠道菌群分布不同性别小鼠的乳酸杆菌总数、肠杆菌总数、肠球菌总数及真菌总数差异均无统计学意义（p>0.05）。提示不同性别的小鼠之间肠道菌群分布无差异，实验有效。3.正常小鼠肠道菌群分布正常小鼠肠道内，肠杆菌所占比例最大，其次为乳酸杆菌，再次为肠球菌及真菌。（二）不同广谱抗生素作用下小鼠肠道菌群分布情况1.抗生素的直接作用随着土霉素剂量的减小，乳酸杆菌总数呈上升趋势，但肠杆菌、肠球菌及真菌随着土霉素剂量的降低一定的降低趋势。土霉素在不同剂量作用下小鼠肠道菌群之间差异无统计学意义（p>0.05）。随着氯霉素剂量的减小，乳酸杆菌总数变化无规律，肠杆菌及肠球菌出现上升趋势，但真菌变化较小。氯霉素低剂量组与对照组间（p=0.011<0.05），氯霉素高剂量组与低剂量组间（p=0.037<0.05），肠球菌总数差异均具有统计学意义。2.抗生素作用后细菌的再定植随着土霉素剂量的减小，乳酸杆菌总数的上升趋势消失，肠杆菌、肠球菌总数出现波动状态，真菌总数呈现降低趋势。提示土霉素在不同剂量作用下小鼠肠道菌群之间差异无统计学意义（p>0.05）。随着氯霉素剂量减小，肠杆菌、肠球菌总数呈现下降趋势，乳酸杆菌变化无规律。氯霉素中剂量组与对照组间（p=0.012<0.05），氯霉素低剂量组与对照组间（p=0.025<0.05），氯霉素高剂量组与中剂量组间（p=0.029<0.05），乳酸杆菌差异具有统计学意义。3.相同剂量不同种类抗生素作用相同剂量不同种类抗生素作用下，无论作用后是否自然恢复，小鼠肠道各菌群之间除抗生素中剂量组两抗生素种类作用下肠球菌总数差异有统计学意义外（p=0.027<0.05），其他剂量组两抗生素间差异均无统计学意义（p>0.05）。4.相同抗生素种类及其剂量作用下不同处理因素对小鼠肠道菌群的影响相同抗生素种类及其剂量作用下，处理因素不同，小鼠肠道菌群的差异经单因素方差分析差异无统计学意义（p>0.05）。（三）致病菌作用下小鼠肠道菌群分布情况在抗生素作用后，给予致病菌灌胃处理，除高剂量土霉素及高剂量大肠埃希菌处理组外，每一组均以肠杆菌所占比例最大。其次为乳酸杆菌，但土霉素低剂量加大肠埃希菌高剂量组、土霉素高剂量加金黄色葡萄球菌高剂量组以肠球菌所占比例居第二。不同抗生素种类及剂量作用后以大肠埃希菌进行处理的各剂量组间各类肠道菌群总数差异无统计学意义（p>0.05）。土霉素高剂量加金黄色葡萄球菌高剂量组与中剂量组间，乳酸杆菌总数差异（p=0.034<0.05），氯霉素高剂量加金黄色葡萄球菌高剂量组与对照组间，乳酸杆菌总数差异（p=0.008<0.05），低剂量组与对照组间，乳酸杆菌总数差异（p=0.026<0.05），低剂量组与对照组间，肠杆菌总数差异（p=0.000<0.05），氯霉素低剂量加金黄色葡萄球菌高剂量组与对照组间，肠杆菌总数差异（p=0.005<0.05），高剂量组与中剂量组间，肠杆菌总数（p=0.007<0.05），上述各组间差异均具有统计学意义。（四）乳酸杆菌拮抗作用对小鼠肠道菌群的影响不同乳酸杆菌剂量在不同处理因素作用后小鼠肠道菌群分布情况各异，加入不同处理因素后小鼠肠道菌群的分布会有一定的影响。部分实验组加入乳酸杆菌后肠道菌群失调有所恢复，多数实验组效果不明显。（五）小鼠肠道菌群分布影响因素影响肠道菌群分布变化的因素因菌种不同而异，其中影响乳酸杆菌总数的主要因素为抗生素种类，其回归方程为y=1.463-1.439x，影响肠杆菌总数的因素按影响作用从大到小分别为终末体重（x1）、设计分组(x2)、致病菌剂量（x3）及实验时间（窝别）（x4），其回归方程为y=-16.164+0.351x1+0.453x2+1.184x3+2.052x4，而肠球菌总数及真菌总数不受本次设计实验因素的影响。结论1.土霉素及氯霉素均可造成小鼠肠道菌群失调，呈现随抗生素剂量减小，乳酸杆菌所占比例增加，而肠杆菌及肠球菌随之比例降低的特征；土霉素对小鼠肠道肠杆菌及肠球菌抑菌作用较强，对乳酸杆菌作用稍弱；氯霉素对肠球菌、乳酸杆菌的影响较土霉素大，随着氯霉素剂量的减小乳酸杆菌呈下降趋势；停药后各类菌群仍出现降低趋势，其慢性毒性作用较大。2.大肠埃希菌或金黄色葡萄球菌作用后对肠道菌群的分布存在差异，氯霉素处理组，致病菌对肠道菌群的影响较明显，尤其金黄色葡萄球菌对乳酸杆菌影响较大，随着氯霉素和金黄色葡萄球菌的减少，乳酸杆菌增加。3.部分实验组，乳酸杆菌对致病菌的拮抗作用对调整和恢复肠道菌群平衡的有一定效果，多数实验组提高乳酸杆菌，减轻菌群失调程度、恢复肠道菌群平衡的作用不显著。4.影响小鼠肠道乳酸杆菌总数的主要因素为抗生素种类，土霉素影响较氯霉素大；影响肠杆菌总数的因素按影响作用从大到小分别为终末体重、实验设计分组、致病菌剂量及动物窝别，终末体重增加，可导致肠道肠杆菌增大；抗生素或致病菌种类及剂量或乳酸杆菌的剂量组合不同可影响肠杆菌的分布；大肠埃希菌或金黄色葡萄球菌的剂量减少可导致肠杆菌的增加；小鼠窝别不同对肠杆菌数量有影响；肠球菌总数及真菌总数未受影响。更多还原

【Abstract】 ObjectiveTo choose standard terramycin and chloromycetin, the pyogenic bacteria as escherichia coli and staphyl-ococcus aureus, and the probiotic of lactobacillus which was easy to be obtained as the research objects, according to the multiple factors and levels orthogonal design on animal experiments, study on weather the effect of pathogenic bacteria was enhance in mice, during attack by pyogenic bacteria after dysbacteriosis molding by antibiotics. Then, make probiotics to adjust the intestinal flora, discuss the efficiency of probiotics to alleviate or restore the intestinal dysbacteriosis, find out the main influence factors in testinal flora distribution, and provide strong scientific evidence of intestinal microecological health in human.MethodsDisposed by intragastric administration with terramycin or chloromycetin according to the multiple factors and levels orthogonal design of each experimental group respectively, keep in3ds. Dispose with pathogenic bacteria7ds after stopping antibiotics4ds. Then stop Id, disposed with lactobacillus for7ds. One day later, the cecum contents were taken out to foster the bacteria with selective media, then,observe the changes of intestinal flora. Results1. Distribution of Intestinal Flora in Normal Mice(1) Distribution of Intestinal Flora in Different GenerationsThe differences were not statistically significant about the total number of lactobacillus, enterobacteria, enterococcus and fungus in normal mice from different experiment(p>0.05). Indicated the distribution of intestinal flora in different generations were no differences, experiments were effective.(2) Distribution of Intestinal Flora in Different GenderThe differences were not statistically significant about the total number of lactobacillus, enterobacteria, enterococcus and fungus in different gender (p>0.05). Indicated the distribution of intestinal flora in different gender were no differences, experiments were effective.(3) Distribution of Intestinal Flora in Normal MiceThe proportion of enterobacteria was the largest, followed by lactobacillus, and there were enterococcus and fungus in normal mice.2. Distribution of Intestinal Flora in Mice Effected by Different Antibiotics(1) Directly Function of AntibioticsThe total number of lactobacillus was increase during the dose decrease of terramycin, but the enterobacteria, enterococcus and fungus were decrease with the dose increase of terramycin. Indicated differences were not statistically significant with intestinal flora in different dose (p>0.05). The lactobacillus was no regular during the dose decrease of chloramphenicol, enterobacteria and enterococcus were increased, but fungus had a little change. The differences were statistically significant with enterococcus, between the low dose group and control group (p=0.011<0.05), and between the higher dose group and the low dose group (p=0.037<0.05).(2) Colonize of Bacteria after Antibiotics EffectThe upward trend of lactobacillus were disappear, during dose decreasing of terramycin, enterobacteria and enterococcus showed decrease tender, lactobacillus changed no regular. The differences were statistically significant about lactobacillus with chloramphenicol, between the middle dose group and control group (p=0.012<0.05), and between the low dose group and control group (p=0.025<0.05),and the high dose group and the middle dose group (p=0.0290.05).(3) Effects of Different Kinds of Antibiotics in Same DoseUnder the effect of different antibiotics in same dose, neither after normal feeding or not, the differences were statistically significant of enterococcus in middle dose of each antibiotics (p=0.0270.05), others were no statistically significant (p>0.05).(4) Influence of Intestinal Flora under Different Process Factors by Antibiotics in Same Species and DosageThe different of intestinal flora under the same species and dosage of antibiotics, dealing with different factors, analysis by ANOVA, no statistically significant was showed (p>0.05).3. Distribution of Intestinal Flora under Pathogenic BacteriumAfter dispose by antibiotics, pathogenic bacterium was given. Except the high dose of terramycin and the high dose of escherichia coli treatment groups, the escherichia coli accounted for the most in each group. Mostly followed by lactobacillus, but enterococcus were the secondary in the group of low dose of terramycin with high dose of escherichia coli, and the group of high dose of terramycin with high dose of staphylococcus aureus. escherichia coli after disposed by different kind and dose of antibiotics (p>0.05). The differences were statistically significant with intestinal flora in some groups. Such as lactobacillus between the groups of high dose of terramycin add high dose of staphylococcus aureus and the middle dose group (p=0.0340.05), and between the group of high dose of chloramphenicol add high dose of staphylococcus aureus and control group (p=0.0080.05), and low dose of staphylococcus aureus and control group (p=0.026<0.05). Enterobacteria between the groups of high dose of chloramphenicol add high dose of staphylococcus aureus and control group(p=0.0000.05), and between the groups of low dose of chloramphenicol add high dose of staphylococcus aureus and control group (p=0.005<0.05), between the high dose of staphylococcus aureus and group middle dose of staphylococcus aureusgroup (p=0.007<0.05).4. Influences of Intestinal Flora in Antagonism Function by LactobacillThere was different distribution after different dispose with different dose of lactobacillus. In some groups, lactobacillus could adjust to intestinal flora, most of the experimental groups showed not so well.5. Influence Factors of Intestinal FloraThe influence factors of intestinal flora were different in different flora. The influence factors of lactobacillus were the kind of antibiotics. The regression equation was y=1.463-1.439x. According the function level from big to small, the influence factors of enterobacterium were final weight (x1), design group(x2), pathogenic bacteria dose(x3) and experimental period or generation(x4). The regression equation was y=-16.164+0.351x1+0.453x2+1.184x3+2.052x4. And enterococcus and fungi were not affected by the experiment factors.Conclusions1. Both Terramycin and Chloromycetin could create dysbacteriosis. The characteristics were showed lactobacilli proportion increase with antibiotic dose reduction, and the proportion of enterobacter and enterococcus in reducing.Terramycin had more restrain effect with enterobacter and enterococcus, but not so strong for lactobacillus. Chloramphenicol had more restrain effect with enterobacter and enterococcus than terramycin, lactobacillus declined with the decrease of the chloramphenicol dose. After discontinuance of chloramphenicol, all kinds of bacteria were decreased, it is may be associated with chronic toxic effect.2. After the effect of escherichia coli and staphylococcus aureus, intestinal flora showed in different distribution.In chloramphenicol group, pathogenic bacterium influenced the intestinal flora obviously. Especially staphylococcus aureus for lactobacillus, it had rebounded with reduce chloromycetin and staphylococcus aureus. 3. In some groups, the antagonism effect to pathogenic bacterium of lactobacillus were useful in adjust and restore intestinal flora balance. Most groups showed no prominent effect to increase lactobacillus, reduce dysbacteriosis and restore intestinal flora balance.4. The influence factors of lactobacillus was the kind of antibiotics, terramycin showed more than chloromycetin. According the function level from big to small, the influence factors of enterobacterium were final weight, design group, pathogenic bacteria dose and experimental period or generation. Final weight increase could lead enterobacterium increase. The combination in different species and dose of antibiotics, athogenic bacteria, or lactobacillus could influence enterobacterium. Reduction the dose of escherichia coli and staphylococcus aureus could lead enterobacterium increase. The difference generation of mice could influence enterobacterium..The enterococcus and fungus were unaffected.更多还原