本文選題：自動哺乳 + 小鼠替代乳�。� 參考：《第三軍醫(yī)大學(xué)》2012年博士論文

【摘要】：抗生素處理是影響嬰兒腸道菌群定植的重要因素之一。臨床上許多廣譜抗生素作為兒科治療的常用藥物，具有減少腸道菌群多樣性等作用。直接利用嬰兒進行腸道菌群研究存在倫理學(xué)限制，利用無菌動物（germ free animal，GF動物）構(gòu)建攜帶嬰兒腸道菌群的人源菌群動物（infant human floral-assocciated animal，IHFA動物）可以為研究抗生素處理對腸道菌群的影響提供了一個有效的模型。IHFA動物模型已有初步的研究和應(yīng)用，但缺乏對嬰兒腸道菌群在無菌動物體內(nèi)的定植規(guī)律的系統(tǒng)研究，因此IHFA動物模型是否適用于抗生素處理對嬰兒腸道菌群的影響未知。此外，由于目前國內(nèi)無菌動物研究和服務(wù)水平較低，限制了IHFA模型的研究和應(yīng)用。因此，本研究擬建立無菌小鼠的自動哺乳技術(shù)，為高效獲得無菌小鼠提供條件；利用無菌小鼠建立IHFA小鼠（infant humanfloral-assocciated mice）模型，研究小鼠年齡和品系因素對嬰兒腸道菌群定植的影響；利用IHFA小鼠研究臨床劑量的阿莫西林處理對嬰兒腸道菌群的影響。 方法 1．為高效制備無菌小鼠，建立乳鼠自動哺乳方法。對3日齡C57BL/6J小鼠實施微創(chuàng)胃瘺手術(shù)（n=48），利用蠕動泵將小鼠替代乳（mouse milk substitute，MMS）通過胃瘺泵入小鼠胃內(nèi)，利用相同日齡的母乳喂養(yǎng)乳鼠作為正常對照組（n=20），觀測其生長發(fā)育利用DGGE指紋圖譜技術(shù)評估其腸道菌群變化。將自動哺乳方法應(yīng)用到無菌剖腹產(chǎn)獲得的新生無菌昆明乳鼠，觀測其生長發(fā)育及成年小鼠的血液生化指標及繁殖能力。 2．為研究嬰兒腸道菌群在無菌小鼠體內(nèi)的定植規(guī)律，將7日齡無菌乳鼠（昆明小鼠n=10；Balb/c小鼠, n=10），接種3月齡健康母乳嬰兒糞便，建立IHFA小鼠模型。利用DGGE指紋圖譜技術(shù)及雙歧桿菌、乳桿菌的特異性引物qRT-PCR檢測postnatal day7（PND7），PND14、PND21的IHFA小鼠腸道菌群，比較品系不同及不同年齡對嬰兒腸道菌群在無菌小鼠體內(nèi)定植的影響。 3．為研究抗生素對嬰兒腸道菌群的影響，將IHFA Balb/c小鼠自PND7至PND21灌胃阿莫西林（100mg/kg，n=10）或等量生理鹽水（n=10）。利用DGGE指紋圖譜技術(shù)及雙歧桿菌、乳桿菌的特異性引物qRT-PCR檢測PND21，PND53IHFA小鼠腸道菌群，研究幼年阿莫西林對IHFA嬰兒腸道菌群的影響及成年后的恢復(fù)情況。 結(jié)果 1．改進了乳鼠的自動哺乳技術(shù)，建立了快速制備無菌小鼠的方法。在自動哺乳條件下，無菌小鼠和普通小鼠均可正常生長發(fā)育。48只自動哺乳11天后，37只C57BL/6J小鼠存活。腸道菌群V3-DGGE圖譜顯示，自動哺乳與母乳喂養(yǎng)組條帶總數(shù)、shannon指數(shù)無顯著差異。然而兩組間少數(shù)DGGE條帶有明顯差異。上述結(jié)果提示，雖然對于3日齡C57BL/6J小鼠能成功進行胃瘺手術(shù)并自動哺乳存活，但自動哺乳會導(dǎo)致腸道菌群發(fā)生改變。成功將乳鼠的自動哺乳方法應(yīng)用于無菌乳鼠，自動哺乳21天后，無菌小鼠存活率可達62.5%，自動哺乳獲得的無菌雌鼠產(chǎn)仔數(shù)量低于母乳喂養(yǎng)獲得的無菌雌鼠（P0.05）。自動哺乳獲得的無菌小鼠和母乳喂養(yǎng)獲得的無菌小鼠的生化指標無顯著差異（P0.05）。 2．通過比較年齡和品系因素對嬰兒腸道菌群定植的影響，系統(tǒng)研究了嬰兒腸道菌群在無菌小鼠體內(nèi)的定植規(guī)律。Balb/c小鼠腸道菌群與人的相似性指數(shù)（Cs）顯著高于昆明小鼠（P0.05）,兩組DGGE條帶有明顯差異，在聚類分析和PCA圖中兩組能很好的分離。不同年齡的IHFA小鼠的DGGE條帶及與人的相似性指數(shù)（Cs）有明顯差異，在聚類分析和PCA圖中兩組能很好的分離。在PND7、14和21，昆明小鼠和Balb/c小鼠體內(nèi)雙歧桿菌和乳桿菌定植數(shù)量無顯著差異（P0.05）。乳桿菌數(shù)量隨小鼠年齡增長而減少，但始終明顯高于人。雙歧桿菌數(shù)量隨小鼠年齡增長而增加，但始終明顯低于人。 3．通過阿莫西林處理組和正常對照組的比較，證明阿莫西林對嬰兒腸道菌群有顯著影響，停止干預(yù)后至53日齡，，仍不能完全恢復(fù)。在PND21的DGGE結(jié)果中，阿莫西林處理組與正常對照組shannon指數(shù)及均勻度差異顯著（P0.05），條帶數(shù)無顯著差異（P0.05），在聚類分析和PCA圖中兩組能很好的分離；在PND53的DGGE結(jié)果中，阿莫西林處理組與正常對照組shannon指數(shù)及豐富度差異顯著（P0.05），均勻度無顯著差異（P0.05），在聚類分析和PCA圖中兩組均不能很好的分離；PND21阿莫西林處理顯著降低腸道菌群中的雙歧桿菌和乳桿菌數(shù)量。在PND53，與正常對照組比較阿莫西林處理組均勻度、乳桿菌數(shù)量及雙歧桿菌數(shù)量均無顯著差異。 結(jié)論 1．通過改進乳鼠自動哺乳技術(shù)，建立了快速制備無菌小鼠的方法。對SPF及無菌乳鼠進行胃瘺手術(shù)和自動哺乳是可行的，乳鼠生長發(fā)育正常，自動哺乳獲得的成年無菌小鼠可正常繁殖獲得子代無菌小鼠，但自動哺乳會導(dǎo)致攜帶正常菌群的乳鼠菌群紊亂。 2．IHFA小鼠能部分模擬嬰兒腸道菌群結(jié)構(gòu)，小鼠年齡和品系因素對嬰兒腸道菌群定植有顯著影響。在制備IHFA小鼠模型時，無菌小鼠的年齡和品系因素均可影響嬰兒腸道菌群定植。嬰兒腸道菌群在無菌小鼠體內(nèi)定植可一定程度的模擬嬰兒腸道菌群，但存在的差異不可忽視（如乳桿菌和雙歧桿菌的數(shù)量差異）。 3．阿莫西林處理可顯著改變IHFA乳鼠的嬰兒腸道菌群結(jié)構(gòu)，且成年后腸道菌群結(jié)構(gòu)未完全恢復(fù)正常。與文獻報道的臨床研究及動物模型研究結(jié)果一致，提示IHFA小鼠是評價抗生素處理對嬰兒腸道菌群影響的有效模型。 創(chuàng)新性 1．本研究首次將乳鼠自動哺乳技術(shù)應(yīng)用于無菌小鼠培育，有利于無菌動物技術(shù)的推廣和應(yīng)用。 2．本研究利用無菌小鼠建立IHFA小鼠模型，首次系統(tǒng)研究小鼠品系及年齡因素對嬰兒腸道菌群定植的影響。 3．本研究首次使用IHFA模型研究抗生素對嬰兒腸道菌群的影響，為評價抗生素對嬰兒腸道菌群的影響提供新模型。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R-332

【共引文獻】

相關(guān)期刊論文 前2條

1 李文霞;曾本華;袁靜;唐歡;魏泓;;GF昆明小鼠主要重量系數(shù)及血液生理指標的測定[J];中國微生態(tài)學(xué)雜志;2011年09期

2 曾本華;唐歡;李文霞;袁靜;鄧人偉;魏泓;;肥胖患者HFA小鼠模型的建立[J];中國微生態(tài)學(xué)雜志;2012年04期

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