摘要
目的采用16S rDNA PCR产物变性梯度凝胶电泳(DGGE)技术探讨早产儿坏死性小肠结肠炎(NEC)肠道菌群多样性及其动态变化。方法以2009年8月至2012年12月在重庆医科大学附属儿童医院和深圳市儿童医院诊断为NEC的早产儿为NEC组,并分为Ⅰ、Ⅱ和Ⅲ期亚组,每个亚组各7例;以2家医院同期住院的非NEC早产儿为对照组,选择与NEC 3个亚组胎龄、日龄、出生体重相匹配者作为对照Ⅰ、Ⅱ和Ⅲ亚组,每个亚组亦各7例。在NEC组和对照组入院后1、3、5、7和9 d时点采集粪便标本,进行DNA扩增和DGGE,对条带进行回收、克隆和测序。分析丰富度S和Shannon指数,通过T-A克隆测序,分析NEC组和对照组肠道细菌种类分布及常见优势菌。结果①入院后1 d时点NEC组Ⅰ、Ⅱ和Ⅲ期亚组Shannon指数分别为(2.01±0.73)、(1.93±0.27)和(1.54±0.36),均显著低于相应的对照亚组[分别为(2.65±0.26)、(2.74±0.16)和(2.56±0.22),P<0.05],且NEC组Ⅲ期亚组Shannon指数显著低于NEC组Ⅰ期和Ⅱ期亚组(P<0.05)。②NEC组和对照组各10例获得全部5个时点的粪便标本,对照组Shannon指数保持较为稳定的水平,NEC组Shannon指数随入院时间的延迟呈升高趋势,于入院9 d时点接近对照组水平。③DGGE胶回收测序结果显示,NEC组与对照组所包含的细菌类别总体差别不大,但NEC组双歧杆菌属、乳酸杆菌属和大肠杆菌属的比例有低于对照组的趋势,而克雷伯菌属和拟杆菌属所占比例有高于对照组的趋势。结论肠道菌落种类和数量的减少,尤其是益生菌的减少,致使克雷伯菌属的相对优势,可能是NEC发病机制中的重要因素。
Objective To analyze the intestinal microbial communiiy diversiiy and its dynamic change in preterm infants with necrotizing enterocolitis （NEC） by 16S rDNA PCR denaturing gradient gel electrophoresis（PCR-DGGE）. Methods The preterm infants diagnosed as NEC during August 2009 to December 2012 in Children＇s Hospital of Chongqing Medical Universiiy and Children＇s Hospital of Shenzhen were recruited as NEC group, and was divided into three subgroups according to stage Ⅰ , Ⅱ and Ⅲ, with 7 infants in each group. The infants without NEC in the two hospitals during the same period were taken as the control group, selected by matching the gestational age, days of age, birih weight with each NEC subgroup and 7 infants in each control subgroup. The stool samples of the NEC infants were collected at 1, 3, 5, 7 and 9 day- point after admission. And the band richness S and Shannon index of them by DNA amplification and DOGE were analyzed. TA Cloning Kit and sequencing were used to investigate the distribution of bacteria and common dominant bacteria in the NEC group and control group. ①The Shannon index of NEC subgroup stage Ⅰ , Ⅱ and Ⅲ on day- 1 after admission was 2.01 ± 0.73, 1.93 ± 0.27 and 1.54 ± 0.36 respectively, which were lower than those of the matched control subgroups （2. 65 ± 0.26, 2.74 ± 0.16, 2.56 ± 0.22 respectively, P 〈0.05） ;and in NEC stage Ⅲ, it was lower than that of NEC stage Ⅰand Ⅱ （P 〈 0.05） . ②In 10 infants in NEC group and 10 infants in control group 5 time points stool samples were obtained; it showed that the Shannon index of the control group kept relatively stable level; and the Shannon index of NEC group elevated gradually with delayed admission time, and about 9 days later, it almost reached the level of the control group,P 〉 0.05. ③The result of molecular eoling and sequencing from DOGE showed thatthe main bacterial types of NEC did not significantly differ from the control group, but the proportions of bifidobacterium, lactobacillus and escherichia coli in NEC group were lower, whlie the proportions of klebsiella and bacteroides sp were higher than that in control group. Conclusion The reduction of intestinal bacteria in types and quantity-, especially the decrease of probioties, may lead to relative advantages for klebsiella, which may be an important factor in the pathogenesis of NEC.
出处
《中国循证儿科杂志》
CSCD
2014年第2期117-121,共5页
Chinese Journal of Evidence Based Pediatrics
基金
国家自然科学基金:81370744
81070513
关键词
坏死性小肠结肠炎
变性梯度凝胶电泳
菌群多样性
益生菌
Necrotizing enterocolitis
Denaturing gel gradient electrophoresis
Microbial conmmnity diversity
Probioties
作者简介
余加林，E-mail：yujialin486@sohu．com
