本文關鍵詞：新生兒腸道菌群分布特征及其影響因素　出處：《上海交通大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： 新生兒 早產兒 腸道菌群 PCR-DGGE 高通量測序

【摘要】：第一部分DGGE分析新生兒期腸道菌群多樣性及影響因素目的:利用PCR-DGGE技術,研究新生兒腸道菌群多樣性的動態(tài)特征,分析影響腸道菌群多樣性的相關因素。方法:采集157例足月兒和72例早產兒生后第3、5、7天及3周的糞便標本,提取細菌總DNA,對其16S r DNA的V3可變區(qū)進行PCR擴增,擴增產物經(jīng)DGGE電泳后計算豐富度和Shannon指數(shù),進行統(tǒng)計學分析。結果:(1)新生兒腸道菌群隨時間動態(tài)變化,3天到3周的豐富度均隨時間增多(6.17±2.23,10.34±2.60,13.04±3.82,13.86±3.81),1周內的Shannon指數(shù)隨時間上升(1.33±0.34,1.74±0.25,1.80±0.31),第3周較第7天的多樣性無明顯變化(P0.05)。第5天豐富度及Shannon指數(shù)明顯高于第3天(P0.001)。(2)出生方式的影響:剖宮產兒生后第3天的豐富度和Shannon指數(shù)(5.63±2.05,1.24±0.35)明顯低于順產兒(6.82±2.28,1.44±0.30,P0.001);第5天Shannon指數(shù)也低于順產兒(P0.05),1周和3周兩組無差異。(3)喂養(yǎng)方式的影響:1周和3周時母乳喂養(yǎng)Shannon指數(shù)(1.75±0.32,1.79±0.32)較非母乳喂養(yǎng)低(1.88±0.29,1.92±0.35,P0.05),豐富度無差異(P0.05)。(4)胎齡的影響:足月兒3、5、7天的豐富度和3、5天的Shannon指數(shù)均明顯高于早產兒。第3周兩組無明顯差異。結論:新生兒期腸道菌群多樣性隨時間呈動態(tài)性變化,3-5天為菌群發(fā)展最迅速時期。剖宮產新生兒早期的腸道菌群多樣性低于順產兒。母乳喂養(yǎng)多樣性低于非母乳喂養(yǎng)。早產兒腸道菌群多樣性低下,定植延遲。第二部分Illumina高通量測序分析胎齡及喂養(yǎng)方式對早產新生兒期腸道菌群分布特征的影響目的:應用Illumina高通量測序技術,研究胎齡及喂養(yǎng)方式對早產兒腸道菌群分布特征的影響。方法:(1)選取剖宮產、母乳及人工喂養(yǎng)的早產兒35名入喂養(yǎng)組;選取剖宮產、人工喂養(yǎng)早產兒22名入胎齡組。(2)將糞便細菌DNA等濃度等比例混合,擴增16Sr DNA V3區(qū),應用Illumina Miseq平臺測序,分析腸道菌群組成特征。結果:(1)早產兒腸道菌群以兩個細菌門類--Firmicutes、Proteobacteria、三個綱--Bacilli、Clostridia、Gammaproteobacteria為主。在科水平以Streptococcaceae、Veillonellaceae、Enterobacteriaceae占主要優(yōu)勢。整個觀察期未檢出Bacteroidetes和Bifidobacterium。(2)喂養(yǎng)方式的影響:兩種喂養(yǎng)方式對主要菌門和菌屬的相對豐度的影響無明顯差異。低豐度水平上,母乳喂養(yǎng)兒比人工喂養(yǎng)兒擁有更豐富的物種,研究發(fā)現(xiàn)Lactobacillus母乳組顯著高于人工喂養(yǎng)組(P0.05)。而Escherichia甚至在人工喂養(yǎng)組未能檢出。另外,在不同時間點兩組菌群結構也存在差異,門水平的差異主要表現(xiàn)在3-5天。(3)胎齡的影響:大胎齡組較小胎齡組擁有更豐富的物種,優(yōu)勢菌屬Veillonella在大胎齡組明顯高于小胎齡組(P0.05)。結論:高通量測序揭示了早產兒腸道菌群特征:更多兼性厭氧菌定植,嚴格厭氧菌定植延遲。胎齡是影響早產兒腸道菌群分布特征的重要因素。喂養(yǎng)方式對主要菌群結構影響不大,人工喂養(yǎng)對部分低豐度菌群的建立存在不良影響。
[Abstract]:Part I DGGE analysis of intestinal microflora diversity and its influencing factors during neonatal period objective: to study the dynamic characteristics of intestinal microflora diversity in neonates by using PCR-DGGE technique. Methods: the fecal specimens of 157 term infants and 72 preterm infants were collected on the 7th and 3rd week after birth to extract total bacterial DNA. The V3 variable region of 16s r DNA was amplified by PCR. The richness and Shannon index were calculated by DGGE electrophoresis. Results the intestinal microflora of newborns changed dynamically with time from 3 days to 3 weeks. The richness of intestinal microflora was 6.17 鹵2.23 鹵10.34 鹵2.60 with the increase of time. The Shannon index of 13.04 鹵3.82v 13.86 鹵3.81g per week increased by 1.33 鹵0.34 鹵1.74 鹵0.25 with time. 1.80 鹵0.31). There was no significant change in diversity between the 3rd week and the 7th day. The richness and Shannon index on the 5th day were significantly higher than that on the 3rd day (P 0.001. 0. 0. 2). The influence of birth style: the richness and Shannon index were 5.63 鹵2.05 on the 3rd day after cesarean section. 1.24 鹵0.35) was significantly lower than that in the normal delivery group (6.82 鹵2.28) 1.44 鹵0.30 (P 0.001); On the 5th day, the Shannon index was also lower than that of the normal birth baby (P0.05). There was no difference between the two groups at 1 and 3 weeks.) the Shannon index of breast-feeding was 1.75 鹵0.32 at 1 and 3 weeks after 1 and 3 weeks. 1.79 鹵0.32) was lower than that of non-breast feeding (1.88 鹵0.291.92 鹵0.35 P0.05). The effect of gestational age on the richness was not different (P0.05. 4): the richness and 3. The Shannon index of 5 days was significantly higher than that of preterm infants. There was no significant difference between the two groups at the third week. Conclusion: the diversity of intestinal flora in neonatal period showed dynamic changes with time. The diversity of intestinal flora in the early stage of cesarean section was lower than that in the normal birth, the diversity of breast-feeding was lower than that of non-breast-feeding, and the diversity of intestinal flora of premature infants was low. Second part Illumina high-throughput sequencing was used to analyze the effects of gestational age and feeding mode on the distribution of intestinal flora in preterm newborn. Objective: to apply Illumina high-throughput sequencing technique. To study the effect of gestational age and feeding methods on the distribution of intestinal flora of premature infants. Methods 35 premature infants with cesarean section, breast milk and artificial feeding were selected into the feeding group. The fecal bacterial DNA concentration was mixed to amplify 16Sr DNA V3 region by cesarean section and artificial feeding of 22 premature infants in gestational age group. Illumina Miseq platform was used to analyze the composition of intestinal flora. Results the intestinal flora of preterm infants was divided into two groups: Firmicutes. Proteobacteria, three classes Bacillian Clostridia. The main Gammaproteobacteria is Streptococcaceaeus veillonellaceae. Enterobacteriaceae was dominant. Bacteroidetes and Bifidobacterium. 2 were not detected during the observation period. The effects of two feeding methods on the relative abundance of the main phylum and genus were not significantly different. Breast-feeding babies are richer in species than artificially fed ones. The study found that the Lactobacillus breast milk group was significantly higher than the artificial feeding group P0. 05, but Escherichia was not even detected in the artificial feeding group. In addition. At different time points, there were differences between the two groups, and the difference of gate level was mainly manifested in the influence of gestational age of 3-5 days. The large gestational age group had more abundant species than the small gestational age group. The dominant Veillonella was significantly higher in the large gestational age group than in the small gestational age group (P 0.05). Conclusion: high throughput sequencing revealed the characteristics of intestinal flora of premature infants: more facultative anaerobes were colonized. Strict anaerobe colonization delay. Gestational age is an important factor affecting the distribution of intestinal flora of premature infants. Feeding mode has little effect on the main flora structure, but artificial feeding has a negative effect on the establishment of some low-abundance microflora.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R722.1

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