建立IHFA小鼠模型研究阿莫西林对婴儿肠道菌群的影响

发布时间：2018-06-06 06:04

本文选题：自动哺乳 + 小鼠替代乳　；参考：《第三军医大学》2012年博士论文

【摘要】：抗生素处理是影响婴儿肠道菌群定植的重要因素之一。临床上许多广谱抗生素作为儿科治疗的常用药物，具有减少肠道菌群多样性等作用。直接利用婴儿进行肠道菌群研究存在伦理学限制，利用无菌动物（germ free animal，GF动物）构建携带婴儿肠道菌群的人源菌群动物（infant human floral-assocciated animal，IHFA动物）可以为研究抗生素处理对肠道菌群的影响提供了一个有效的模型。IHFA动物模型已有初步的研究和应用，但缺乏对婴儿肠道菌群在无菌动物体内的定植规律的系统研究，因此IHFA动物模型是否适用于抗生素处理对婴儿肠道菌群的影响未知。此外，由于目前国内无菌动物研究和服务水平较低，限制了IHFA模型的研究和应用。因此，本研究拟建立无菌小鼠的自动哺乳技术，为高效获得无菌小鼠提供条件；利用无菌小鼠建立IHFA小鼠（infant humanfloral-assocciated mice）模型，研究小鼠年龄和品系因素对婴儿肠道菌群定植的影响；利用IHFA小鼠研究临床剂量的阿莫西林处理对婴儿肠道菌群的影响。方法 1．为高效制备无菌小鼠，建立乳鼠自动哺乳方法。对3日龄C57BL/6J小鼠实施微创胃瘘手术（n=48），利用蠕动泵将小鼠替代乳（mouse milk substitute，MMS）通过胃瘘泵入小鼠胃内，利用相同日龄的母乳喂养乳鼠作为正常对照组（n=20），观测其生长发育利用DGGE指纹图谱技术评估其肠道菌群变化。将自动哺乳方法应用到无菌剖腹产获得的新生无菌昆明乳鼠，观测其生长发育及成年小鼠的血液生化指标及繁殖能力。 2．为研究婴儿肠道菌群在无菌小鼠体内的定植规律，将7日龄无菌乳鼠（昆明小鼠n=10；Balb/c小鼠, n=10），接种3月龄健康母乳婴儿粪便，建立IHFA小鼠模型。利用DGGE指纹图谱技术及双歧杆菌、乳杆菌的特异性引物qRT-PCR检测postnatal day7（PND7），PND14、PND21的IHFA小鼠肠道菌群，比较品系不同及不同年龄对婴儿肠道菌群在无菌小鼠体内定植的影响。 3．为研究抗生素对婴儿肠道菌群的影响，将IHFA Balb/c小鼠自PND7至PND21灌胃阿莫西林（100mg/kg，n=10）或等量生理盐水（n=10）。利用DGGE指纹图谱技术及双歧杆菌、乳杆菌的特异性引物qRT-PCR检测PND21，PND53IHFA小鼠肠道菌群，研究幼年阿莫西林对IHFA婴儿肠道菌群的影响及成年后的恢复情况。结果 1．改进了乳鼠的自动哺乳技术，建立了快速制备无菌小鼠的方法。在自动哺乳条件下，无菌小鼠和普通小鼠均可正常生长发育。48只自动哺乳11天后，37只C57BL/6J小鼠存活。肠道菌群V3-DGGE图谱显示，自动哺乳与母乳喂养组条带总数、shannon指数无显著差异。然而两组间少数DGGE条带有明显差异。上述结果提示，虽然对于3日龄C57BL/6J小鼠能成功进行胃瘘手术并自动哺乳存活，但自动哺乳会导致肠道菌群发生改变。成功将乳鼠的自动哺乳方法应用于无菌乳鼠，自动哺乳21天后，无菌小鼠存活率可达62.5%，自动哺乳获得的无菌雌鼠产仔数量低于母乳喂养获得的无菌雌鼠（P0.05）。自动哺乳获得的无菌小鼠和母乳喂养获得的无菌小鼠的生化指标无显著差异（P0.05）。 2．通过比较年龄和品系因素对婴儿肠道菌群定植的影响，系统研究了婴儿肠道菌群在无菌小鼠体内的定植规律。Balb/c小鼠肠道菌群与人的相似性指数（Cs）显著高于昆明小鼠（P0.05）,两组DGGE条带有明显差异，在聚类分析和PCA图中两组能很好的分离。不同年龄的IHFA小鼠的DGGE条带及与人的相似性指数（Cs）有明显差异，在聚类分析和PCA图中两组能很好的分离。在PND7、14和21，昆明小鼠和Balb/c小鼠体内双歧杆菌和乳杆菌定植数量无显著差异（P0.05）。乳杆菌数量随小鼠年龄增长而减少，但始终明显高于人。双歧杆菌数量随小鼠年龄增长而增加，但始终明显低于人。 3．通过阿莫西林处理组和正常对照组的比较，证明阿莫西林对婴儿肠道菌群有显著影响，停止干预后至53日龄，，仍不能完全恢复。在PND21的DGGE结果中，阿莫西林处理组与正常对照组shannon指数及均匀度差异显著（P0.05），条带数无显著差异（P0.05），在聚类分析和PCA图中两组能很好的分离；在PND53的DGGE结果中，阿莫西林处理组与正常对照组shannon指数及丰富度差异显著（P0.05），均匀度无显著差异（P0.05），在聚类分析和PCA图中两组均不能很好的分离；PND21阿莫西林处理显著降低肠道菌群中的双歧杆菌和乳杆菌数量。在PND53，与正常对照组比较阿莫西林处理组均匀度、乳杆菌数量及双歧杆菌数量均无显著差异。结论 1．通过改进乳鼠自动哺乳技术，建立了快速制备无菌小鼠的方法。对SPF及无菌乳鼠进行胃瘘手术和自动哺乳是可行的，乳鼠生长发育正常，自动哺乳获得的成年无菌小鼠可正常繁殖获得子代无菌小鼠，但自动哺乳会导致携带正常菌群的乳鼠菌群紊乱。 2．IHFA小鼠能部分模拟婴儿肠道菌群结构，小鼠年龄和品系因素对婴儿肠道菌群定植有显著影响。在制备IHFA小鼠模型时，无菌小鼠的年龄和品系因素均可影响婴儿肠道菌群定植。婴儿肠道菌群在无菌小鼠体内定植可一定程度的模拟婴儿肠道菌群，但存在的差异不可忽视（如乳杆菌和双歧杆菌的数量差异）。 3．阿莫西林处理可显著改变IHFA乳鼠的婴儿肠道菌群结构，且成年后肠道菌群结构未完全恢复正常。与文献报道的临床研究及动物模型研究结果一致，提示IHFA小鼠是评价抗生素处理对婴儿肠道菌群影响的有效模型。创新性 1．本研究首次将乳鼠自动哺乳技术应用于无菌小鼠培育，有利于无菌动物技术的推广和应用。 2．本研究利用无菌小鼠建立IHFA小鼠模型，首次系统研究小鼠品系及年龄因素对婴儿肠道菌群定植的影响。 3．本研究首次使用IHFA模型研究抗生素对婴儿肠道菌群的影响，为评价抗生素对婴儿肠道菌群的影响提供新模型。
[Abstract]:Antibiotic treatment is one of the important factors affecting the colonization of intestinal flora in infants. Many broad-spectrum antibiotics, as commonly used drugs for pediatric treatment, have the effect of reducing the diversity of intestinal flora. There are ethical restrictions on the direct use of infants for intestinal microflora study, and the use of germ free animal, GF animals to build and carry Infant human floral-assocciated animal (IHFA animal) can provide an effective model.IHFA animal model for the study of the effect of antibiotic treatment on intestinal flora, but there is a lack of system for the colonization of infantile intestinal flora in aseptic animals. Therefore, whether the IHFA animal model is applicable to the effect of antibiotic treatment on the intestinal flora of infants is unknown. In addition, due to the low level of research and service in the domestic aseptic animals, the research and application of the IHFA model are limited. Therefore, this study intends to establish the automatic breast-feeding technique for aseptic mice to provide the conditions for efficient aseptic mice. A IHFA mouse (infant humanfloral-assocciated mice) model was used to study the effect of age and strain on the intestinal colonization of infantile intestinal flora, and the effect of amoxicillin on the intestinal flora of infants was studied by using IHFA mice.
Method
1. for the efficient preparation of sterile mice and the establishment of milk mouse automatic lactation method. The 3 day old C57BL/6J mice were treated with minimally invasive gastric fistula operation (n=48), and the mouse replacement milk (mouse milk substitute, MMS) was pumped into the stomach through the gastric fistula by the peristaltic pump, and the milk mice were fed with the same age of breast milk as the normal control group (n=20), and the growth and development of the mice were observed. The DGGE fingerprinting technique was used to evaluate the changes in the intestinal flora. The automatic lactation method was applied to the newborn aseptic Kunming milk rats obtained by aseptic caesarean section, and the growth and development of the mice were observed and the blood biochemical indexes and reproductive capacity of the adult mice were observed.
2. in order to study the colonization of infantile intestinal flora in aseptic mice, the 7 day old sterile mice (Kunming mice n=10; Balb/c mice, n=10) were inoculated with 3 month old healthy breast milk, and the IHFA mouse model was established. The DGGE fingerprinting technique and bifidobacteria and specific primers qRT-PCR of lactobacillus were used to detect postnatal Day7 (PND7) and PND. PND 14, the intestinal flora of PND21 IHFA mice was compared with the effects of different strains and ages on the colonization of intestinal flora in aseptic mice.
3. in order to study the effect of antibiotics on the intestinal flora of infants, IHFA Balb/c mice were intragastric to amoxicillin (100mg/kg, n=10) or equal amount of normal saline (n=10) from PND7 to PND21. Using DGGE fingerprinting technology and bifidobacteria, specific primers of Lactobacillus, qRT-PCR detected PND21, PND53IHFA mouse intestinal flora, and studied juvenile amoxicillin against IHFA. The influence of infantile intestinal flora and the recovery after adult.
Result
1. improved the automatic lactation technique of milk mice and established a method for rapid preparation of aseptic mice. Under the condition of automatic lactation, the aseptic mice and ordinary mice could grow and develop normally for 11 days, and 37 C57BL/6J mice survived. The V3-DGGE map of the intestinal flora showed that the total number of spontaneous lactation and breastfeeding group, Shannon finger However, there were no significant differences in the number of DGGE groups. The results suggested that the automatic lactation could lead to a change in the intestinal flora of the 3 day old C57BL/6J mice, while the automatic lactating method was successfully applied to sterile mice for 21 days after 21 days of nursing. The survival rate of the bacteria mice could reach 62.5%. The number of sterile female rats obtained by automatic lactation was lower than that of the sterile female mice (P0.05). There was no significant difference in the biochemical indexes between the aseptic mice and the aseptic mice obtained by breast feeding (P0.05).
2. by comparing the influence of age and strain on the colonization of intestinal flora in infants, it was studied that the intestinal microflora in the.Balb/c mice was significantly higher than that of Kunming mice (P0.05), and the two groups of DGGE bands were significantly different. The two groups in the cluster analysis and the PCA map could be very different. Good separation. The DGGE bands of IHFA mice of different ages and the human similarity index (Cs) have obvious differences. In the cluster analysis and the PCA map, two groups can be well separated. In PND7,14 and 21, the number of Bifidobacterium and Lactobacillus in Kunming and Balb/c mice has no significant difference (P0.05). The number of Lactobacillus increases with the age of mice. The number of bifidobacteria increased with the age of mice, but it was always lower than that of human beings.
3. compared with the amoxicillin treatment group and the normal control group, it was proved that amoxicillin had a significant influence on the intestinal flora of the infants. After stopping the intervention to 53 days old, the amoxicillin was still not completely restored. In the DGGE results of PND21, the Shannon index and the uniformity of amoxicillin treated group were significantly different (P0.05), and there was no significant difference in the number of bands (P0 .05), in the cluster analysis and the PCA map, two groups were well separated. In the DGGE results of PND53, the Shannon index and the richness of the amoxicillin treated group were significantly different (P0.05), and the evenness was not significantly different (P0.05). In the cluster analysis and PCA map, the two groups were not well separated; PND21 amoxicillin significantly reduced the intestine. The number of bifidobacteria and Lactobacillus in the microorganism group. In PND53, there was no significant difference in the homogeneity of amoxicillin treated group compared with the normal control group, and the number of Lactobacillus and the number of bifidobacteria were not significant.
conclusion
1. the method of rapid preparation of aseptic mice was established by improving milk mouse automatic lactation technology. It was feasible to perform gastric fistula operation and automatic breast-feeding for SPF and aseptic milk mice. The growth and development of suckling mice was normal. The adult sterile mice obtained by automatic breast-feeding could reproduce aseptic mice normally, but the automatic breast-feeding would lead to the carrying of normal flora. A disorder of the Lactobacillus flora.
The 2.IHFA mice can partially simulate the structure of the infantile intestinal flora, and the age and strain factors of the mice have a significant influence on the colonization of the intestinal flora. In the preparation of the IHFA mouse model, the age and strain factors of the sterile mice can affect the colonization of the infantile intestinal flora. The difference of intestinal flora is not negligible, such as the quantity difference between Lactobacillus and bifidobacteria.
3. amoxicillin can significantly change the intestinal microflora structure in the infants of IHFA milk mice, and the intestinal microflora structure after adult is not fully restored to normal. It is consistent with the reports of clinical and animal model studies, suggesting that IHFA mice are an effective model to evaluate the effect of antibiotic treatment on the intestinal flora of infants.
Innovativeness
1. this study is the first to apply the technology of suckling mouse automatic lactation to the cultivation of aseptic mice, which is conducive to the popularization and application of aseptic animal technology.
2. in this study, a IHFA mouse model was established using sterile mice. The effects of strain and age on the colonization of intestinal flora in infants were systematically studied for the first time.
3. this study used the IHFA model for the first time to study the effects of antibiotics on the intestinal flora in infants, and to provide a new model for evaluating the effects of antibiotics on the intestinal flora in infants.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-332

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