苯扎氯铵制作的大鼠先天性巨结肠模型肠道菌群变化的研究  

Change of colonic microbiome existed in benzalkonium chloride-treated Hirschsprung's disease rat model

作　　者：余辉[1] 田东浩 许文耀 赵育樱 杨薇粒 潘伟康[1] 郑百俊[1] 高亚[1] Yu Hui;Tian Donghao;Xu Wenyao;Zhao Yuying;Yang Weili;Pan Weikang;Zheng Baijun;Gao Ya(Department of Pediatric Surgery,Second Affiliated Hospital,Xi'an Jiaotong University,Xi'an 710004,China)

机构地区：[1]西安交通大学第二附属医院小儿外科,710004

出　　处：《中华小儿外科杂志》2021年第2期152-158,共7页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(82071692、81741096、81770513、81701501);陕西省青年科技新星(2019KJXX-044);陕西省自然科学基金(2020JM-407);西安交通大学第二附属医院科技新苗项目(RC(XM)201703)。

摘　　要：目的验证苯扎氯铵制作的大鼠先天性巨结肠模型是否存在肠道菌群变化并探索其规律。方法按随机数字表法将18只200 g左右SPF级Sprague-Dawley(SD)雌鼠随机划分为实验组和对照组,每组各9只,实验组采用0.1%-30 min苯扎氯铵液制作先天性巨结肠模型,对照组用无菌生理盐水处理。分别于术后2、3、4周采集上述模型鼠狭窄段、扩张段以及对照组肠段粪菌,提取粪菌DNA。16S rRNA V3-V4区扩增后,利用Illumina MiSeq测序平台检测,并进行基于OUTs的聚类分析,门水平、属水平菌群相对丰度及多样性分析。以α=0.05为显著性水平,采用One-way ANOVA和Bonferroni校正的事后多重检验,或Kruskal-Wallis法进行差异性分析。结果实验组狭窄段、扩张段以及对照组肠道菌群总OUTs分别为876.33±27.21、913.00±43.51和6193.33±1.53,特有OUTs分别为52.00±16.46、64.67±3.78、5375.33±60.12,各组上述指标与对照组比较,差异均有统计学意义(P均<0.0001)。实验组狭窄段、扩张段与对照组菌群ACE指数分别为1040.15±44.2、959.30±27.95和11923.52±36.66,Chao1指数分别为1087.47±130.35、946.63±29.56和10133.08±69.88,各组上述指标与对照组比较,差异均有统计学意义(P均<0.0001)。实验组狭窄段、扩张段以及对照组菌群Shannon值分别为6.46±0.52、6.50±0.37和8.18±0.01,各组与对照组比较,差异均有统计学意义(P均<0.05)。对照组及实验组狭窄段、扩张段菌群门水平相对丰度较多的是厚壁菌门[(38.46±0.03)%、(72.68±6.15)%、(63.98±7.22)%]、拟杆菌门[(26.76±0.05)%、(24.91±5.98)%、(33.17±6.92)%]和变形菌门[(32.15±0.09)%、(0.70±0.30)%、(0.56±0.14)%],除拟杆菌门外,各组与对照组比较,差异均有统计学意义(P均<0.05)。实验组与对照组菌群属水平相对丰度前10的菌群比较,差异亦有统计学意义(P均<0.05)。结论苯扎氯铵制作的大鼠先天性巨结肠模型存在肠道菌群变化。Objective To verify whether the change of intestinal microbiome existed in rat model of benzalkonium chloride-treated Hirschsprung's disease and to explore its recurring patterns.Methods Eighteen SPF Sprague-Dawley(SD)female rats about 200 g were randomly divided into experimental and control groups(n=9 each).Serosal application of 0.1%-30 min benzalkonium chloride was applied for establishing a model of Hirschsprung's disease in experimental group while control group received only sterile saline.Feces in stricture and expansion of both groups were collected at Week 2/3/4 postoperatively.And fecal DNA was extracted.The v3-v4 region of bacterial 16S rRNA was selected for gene amplication and sequencing using Illumina sequencing platform for OTUs,the relative abundance differences at genus/phylum level as well as diversity analysis.One-way ANOVA and Bonferroni corrected multiple tests or Kruskal-Wallis method were employed for difference analysis withα=0.05 as the significance level.Results The fecal OTUs in stricture/expansion of experimental and control group were 876.33±27.21,913.00±43.51 and 6193.33±1.53.And the specific OUTs were 52.00±16.46,64.67±3.78 and 5375.33±60.12.The differences had statistical significance(all P<0.0001).Significant differences in ACE/Chao1 existed between stricture/expansion of experimental and control groups(1040.15±44.2 vs.959.30±27.95 vs.11923.52±36.66,1087.47±130.35 vs.946.63±29.56 vs.10133.08±69.88,all P<0.0001).The Shannon values of stricture/expansion of experimental and control groups were(6.46±0.52,6.50±0.37&8.18±0.01).And there were significant differences(all P<0.05).Firmicutes,Bacteroidetes and Proteobacteria were the most three abundant species at the phylum level in stricture/expansion of experimental and control groups.The relative abundance of the above species was different(38.46%±0.03%,26.76%±0.05%,32.15%±0.09%vs.72.68%±6.15%,24.91%±5.98%,0.70%±0.30%vs.63.98%±7.22%,33.17%±6.92%,0.56%±0.14%).Except for Bacteroidetes,the differences were statist

关 键 词：HIRSCHSPRUNG病 动物模型 DNA测序 

分 类 号：R725.7[医药卫生—儿科]