基于16S rDNA测序分析脓毒症大鼠早期肠道微生态的变化  

Analysis of the changes in intestinal microecology in the early stage of sepsis rat based on 16S rDNA sequencing

作　　者：李弘毅 翟瑞卿 梁火燕 朱彦辉 闫岩 王诚扬 丁显飞 宋高飞 孙同文[1] Li Hongyi;Zhai Ruiqing;Liang Huoyan;Zhu Yanhui;Yan Yan;Wang Chengyang;Ding Xianfei;Song Gaofei;Sun Tongwen(Department of General Intensive Care Unit,the First Affiliated Hospital of Zhengzhou University,Henan Provincial Key Laboratory of Critical Medicine,Henan Provincial Engineering Research Center for Critical Care Medicine,Zhengzhou Key Laboratory of Sepsis,Zhengzhou 450052,Henan,China;College of Bioinformatics Science and Technology,Harbin Medical University,Harbin 150081,Heilongjiang,China)

机构地区：[1]郑州大学第一附属医院综合ICU,河南省重症医学重点实验室,河南省重症医学工程研究中心,郑州市脓毒症重点实验室,郑州450052 [2]哈尔滨医科大学生物信息科学与技术学院,黑龙江哈尔滨150081

出　　处：《中华危重病急救医学》2022年第1期28-34,共7页Chinese Critical Care Medicine

基　　金：国家自然科学基金(U2004110,82172129);河南省医学科技攻关计划重大项目(SBGJ202101015)。

摘　　要：目的通过16S rDNA测序技术探究脓毒症大鼠早期肠道微生态变化。方法将60只雄性SD大鼠按随机数字表法分为盲肠结扎穿孔术(CLP)组和假手术组(Sham组),每组30只。CLP组采用CLP法制备脓毒症大鼠模型;Sham组只开腹但不进行盲肠结扎穿孔。术后24 h每组取8只大鼠肠道粪便及腹主动脉血,其余大鼠用于观察7 d存活情况。采用酶联免疫吸附试验(ELISA)检测血清肿瘤坏死因子-α(TNF-α)水平;对粪便样本进行16S rDNA测序,利用序列对比和聚类后获得的操作分类单元(OTU)信息进行多样性分析(α多样性和β多样性)、主坐标分析及线性判别分析效应量分析(LEfSe),观察脓毒症大鼠早期肠道微生态的变化并挖掘标志性菌群。结果制模后24 h,CLP组大鼠出现呼吸急促、毛发散乱等表现,且血清TNF-α水平较Sham组显著升高(ng/L:43.95±9.05比11.08±3.27,P<0.01);制模后7 d,Sham组大鼠全部存活,而CLP组大鼠累积生存率仅31.82%。多样性分析显示,Sham组与CLP组α多样性指标比较差异无统计学意义〔物种数量(个):520.00±52.15比492.25±86.61,Chao1丰富度估计量:707.25±65.69比668.93±96.50,Shannon指数:5.74±0.42比5.79±0.91,Simpson指数:0.93±0.03比0.94±0.05,均P>0.05〕;β多样性分析显示,无论是否根据OTU加权,组间差异均大于组内差异(丰度加权矩阵:R=0.23,P=0.04;丰度不加权矩阵:R=0.32,P=0.01)。门水平差异菌群分析显示,与Sham组相比,CLP组变形菌门和TM7菌门丰度显著升高〔18.100%(15.271%,26.665%)比6.974%(2.854%,9.764%),0.125%(0.027%,0.159%)%比0.018%(0.008%,0.021%),均P<0.05〕;在属水平上,CLP组螺杆菌属、钌杆菌属、颗粒链菌属、梭菌属ⅩⅧ等机会致病菌的丰度较Sham组显著升高〔5.090%(1.812%,6.598%)比0.083%(0.034%,0.198%),0.244%(0.116%,0.330%)比0.016%(0.008%,0.029%),0.006%(0.003%,0.010%)比0.001%(0%,0.003%),0.094%(0.035%,0.430%)比0.007%(0.003%,0.030%),均P<0.05〕,而拟普雷沃菌属、罗姆布茨菌属等益生Objective To investigate the changes of intestinal microecology in the early stage of sepsis rat model by 16S rDNA sequencing.Methods Sixty male Sprague-Dawley(SD)rats were randomly divided into cecal ligation and puncture(CLP)group and sham operation group(Sham group),with 30 rats in each group.In the CLP group,sepsis rat model was reproduced by CLP method;the rats in the Sham group only underwent laparotomy without CLP.At 24 hours after the operation,the intestinal feces and serum samples of 8 rats in each group were collected.The survival rate of the rest rats was observed until the 7th day.The level of serum tumor necrosis factor-α(TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA).Intestinal feces were sequenced by 16S rDNA sequencing technology.The operational taxonomic unit(OTU)data obtained after sequence comparison and clustering was used forαdiversity andβdiversity analysis,principal coordinate analysis and linear discriminant analysis effect size analysis(LEfSe)to observe the changes of intestinal microecology in early sepsis rats and excavate the marker flora.Results At 24 hours after the reproduction of the model,the rats in the CLP group showed shortness of breath,scattered hair and other manifestations,and the level of serum TNF-αincreased significantly as compared with that in the Sham group(ng/L:43.95±9.05 vs.11.08±3.27,P<0.01).On the 7th day after modeling,the cumulative survival rate of the Sham group was 100%,while that of the CLP group was 31.82%.Diversity analysis showed that there was no significant difference inαdiversity parameter between the Sham group and the CLP group(number of species:520.00±52.15 vs.492.25±86.61,Chao1 richness estimator:707.25±65.69 vs.668.93±96.50,Shannon index:5.74±0.42 vs.5.79±0.91,Simpson index:0.93±0.03 vs.0.94±0.05,all P>0.05).However,theβdiversity analysis showed that the difference between groups was greater than that within groups whether weighted according to OTU or not(abundance weighted matrix:R=0.23,P=0.04;abundance unweighte

关 键 词：肠道菌群 脓毒症 盲肠结扎穿孔术 16S rDNA测序 

分 类 号：R459.7[医药卫生—急诊医学]