构建HFA动物模型研究粪便菌群在CMPA发生中的作用

发布时间：2018-04-09 10:37

本文选题：粪便菌群　切入点：牛奶蛋白过敏　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的:将牛奶蛋白过敏婴儿和正常婴儿的粪便菌群分别移植给无菌小鼠后,构建菌群人源化小鼠模型,观察移植小鼠对牛奶蛋白的反应,探究粪便菌群在牛奶蛋白过敏发生中的作用。方法:收集重庆医科大学附属儿童医院消化专科临床诊断为牛奶蛋白过敏(cow’milk protein allergy,CMPA)且以胃肠道表现为主的婴儿粪便(I-CMPA组),同时收集该院儿保科体检的、无胃肠道症状且无过敏性疾病的正常婴儿粪便(I-NC组),分别移植给无菌小鼠后为过敏移植小鼠组(M-CMPA组)和正常移植小鼠组(M-NC组),两组移植小鼠均用婴儿配方奶粉喂养。14天后,用Illumina测序技术检测粪便菌群的定植规律和变化情况、观察移植小鼠的临床表现、HE染色观察空肠组织的病理改变、流式细胞术检测脾脏细胞中CD4+CD25+Foxp3+调节性T细胞比例变化。结果:(1)Illumina测序结果分析显示,供体粪菌间的优势菌不同,且I-CMPA组菌属丰富度和多样性均较I-NC组低。粪菌移植14天后,I-CMPA组中的重要组分能在无菌小鼠肠道内较好维持;两组移植小鼠的菌群结构差异明显,菌属水平主成分分析(PCA)图和层级聚类分析图中两组均能独立分开。组间差异物种分析结果显示:I-CMPA组与M-CMPA组中双歧杆菌属的比例均高于其对照组;(2)用婴儿配方奶粉喂养后,M-CMPA组出现肛门红肿、粪便变稀等表现,而M-NC组没有出现类似的表现;(3)HE染色可见M-CMPA组较M-NC组出现明显的空肠绒毛上皮细胞局灶性脱落,绒毛连续性破坏和炎症细胞浸润;(4)流式细胞分析结果显示,M-CMPA组脾脏CD4+CD25+Foxp3+调节性T细胞比例较M-NC组降低。结论:(1)HFA小鼠能较好地模拟CMPA婴儿的肠道微生物区系,该HFA模型为粪便菌群与CMPA的关系及机制研究提供了新的选择;(2)粪便菌群的差异使移植小鼠对牛奶蛋白的反应不同,粪便菌群的变化可能是CMPA发生的始动因素,食物过敏的敏感性可通过粪菌移植的方式在不同种属的宿主间水平传递;(3)双歧杆菌属中的某些特定菌种可能参与CMPA发生。
[Abstract]:Objective: to establish a humanized mouse model of milk protein allergic infant and normal infant fecal microflora, and to observe the reaction of the transplanted mice to milk protein.To explore the role of fecal microflora in the development of milk protein allergy.Methods: the feces of infants with gastrointestinal symptoms were collected and collected from the digestive department of Chongqing Medical University affiliated Children's Hospital, which were diagnosed as milk protein allergyra (CMPA) and were collected from the pediatric care department of Chongqing Medical University for physical examination at the same time.Normal infant feces with no gastrointestinal symptoms and no allergic diseases were treated with I-NC group, which were fed with infantile formula for .14 days after inoculation with anaphylactic mice (group M CMPA) and normal group (group M NC).The colonization and changes of fecal flora were detected by Illumina sequencing technique. The clinical manifestations of transplanted mice were observed. The pathological changes of jejunum tissue were observed by HE staining. The percentage of CD4 CD25 Foxp3 regulatory T cells in spleen cells was detected by flow cytometry.Results the sequence analysis of Illumina showed that the dominant bacteria in donor feces were different, and the richness and diversity of bacteria in I-CMPA group were lower than those in I-NC group.After 14 days of fecal bacteria transplantation, the important components of I-CMPA group could be maintained in the intestinal tract of aseptic mice, and there were significant differences between the two groups in the microflora structure of the transplanted mice, and the two groups could be separated independently from each other by PCAG and hierarchical cluster analysis.The results of species analysis showed that the proportion of Bifidobacterium in the two groups was higher than that in the control group (P < 0.05). The anus redness and fecal thinning were observed in the M-CMPA group after infantile formula was fed, and the rate of bifidobacterium in the two groups was higher than that in the control group (P < 0.05).However, there was no similar appearance in M-NC group. It was found that the focal exfoliation of chorionic villi in M-CMPA group was more obvious than that in M-NC group.The results of flow cytometry showed that the percentage of CD4 CD25 Foxp3 regulatory T cells in spleen of M-CMPA group was lower than that of M-NC group.Conclusion the microflora of the intestinal tract of CMPA infants can be well simulated by the CMPA mice. The HFA model provides a new choice for the study of the relationship and mechanism between fecal microflora and CMPA) the difference of faecal microflora makes the reaction to milk protein of transplanted mice different.The change of faecal flora may be the initiator of CMPA, and the sensitivity of food allergy can be transferred between hosts of different species by fecal bacteria transplantation. Some specific strains of Bifidobacterium may be involved in the occurrence of CMPA.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.9

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