PCR-DGGE对新生儿坏死性小肠结肠炎肠道菌落结构其动态性研究

发布时间：2018-05-02 22:00

  本文选题：新生儿坏死性小肠结肠炎 + 变形梯度凝胶电泳　； 参考：《重庆医科大学》2012年硕士论文

【摘要】：目的 新生儿坏死性小肠结肠炎（NEC, neonatal necrotizingenterocolitis）是新生儿特别是早产儿的急腹症，临床以腹胀、呕吐、便血、严重休克为表现，X线以肠壁囊样积气为特征。本病在国内的病死率高，尤其是早产儿,严重影响新生儿健康。NEC是多因素的疾病，细菌感染是其中一个不可缺少的致病因素。有研究表明，NEC未被发现在无菌的动物模型上,说明了无菌无NEC。有假说表明新生儿肠道异常细菌的定植是NEC发生的一个重要因素。但目前国内外对NEC肠道细菌感染的细菌成分和动态性变化并无清晰的了解。本实验采用PCR结合DGGE来观察NEC肠道微生态成分和动态变化，为NEC疾病的发病机制和临床防治提供细菌学理论基础。 材料与方法 从重庆市儿童医院新生儿病房2009年8月份-2009年12月份共收治32例，其中NEC组和对照组各16例。NEC组根据实用儿科学诊断标准收取，每例均有x片（+）及大便隐血（+）。根据国际Walsh andKliegman的BELL对于NEC疾病的分期标准,均为Ⅰ期(轻度)。对照组根据基础疾病以及临床特征：性别，胎龄，生产方式，喂养方式，出生体重，抗生素使用时间来尽量匹配。取其新鲜大便，提肠道细菌总DNA，用16s rDNA V3可变区引物进行聚合酶链反应（PCR，polymerase chain reaction）扩增，然后变性梯度凝胶电泳（DGGE，denaturing gradient gel electrophoresis），割胶回收细菌DNA测序，进行肠道细菌多样性分析包括丰富度（richness，S）和Shannon index（H′）指数，序列比对分析。 结果 变形梯度凝胶电泳结果显示：13/16（即81.25%）的NEC组跟对照组比较，肠道细菌菌落的Shannon指数相对较高(NEC组vs对照组,p0.05)，分布不均，波动明显，细菌的多样性高于对照组，，细菌菌落多样性特征存在一定的差异。NEC组的Shannon指数在发病初期明显增多,随着疾病的治疗与恢复，呈先降后升的趋势。DGGE胶回收测序显示新生儿肠道优势菌群以肺炎克雷伯杆菌属，大肠埃希氏菌属，肠球菌属为主，NEC组与对照组种类没有明显区别，存在数量上的差异。3/16（18.75%）趋势不明显，shannon index差异不大。 结论 实验表明，NEC患儿的肠道菌群失衡可能是NEC发病的肠道细菌感染的重要因素。
[Abstract]:Purpose Neonatal necrotizing enterocolitis (neonatal necrotizing enterocolitis) is an acute abdomen of newborns, especially premature infants. It is characterized by abdominal distension, vomiting, hematochezia and severe shock. The mortality of this disease is high in China, especially in premature infants. NEC is a multifactor disease, and bacterial infection is one of the indispensable pathogenic factors. Some studies have shown that NEC was not found in sterile animal models, indicating aseptic NECs. It is hypothesized that the colonization of abnormal intestinal bacteria in neonates is an important factor in the development of NEC. However, there is no clear understanding of the changes of bacterial composition and dynamics of NEC enterobacterial infection at home and abroad. In this study, PCR and DGGE were used to observe the microecological composition and dynamic changes of intestinal tract of NEC, which provided a theoretical basis for the pathogenesis of NEC disease and its clinical prevention and treatment. Materials and methods From August 2009 to December 2009, 32 cases were treated in the neonatal ward of Chongqing Children's Hospital. Among them, 16 cases in the NEC group and 16 cases in the control group were collected according to the diagnostic criteria of practical pediatrics. Each case had X ray and fecal occult blood. According to the international standard of BELL of Walsh andKliegman for staging of NEC disease, all of them were stage 鈪

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