重症急性胰腺炎不同病程的细菌移位及肠道菌群组成结构的变化研究

Changes of bacterial translocation and composition of intestinal flora in different stages of severe acute pancreatitis

目的探究重症急性胰腺炎(severe acte pancreatitis,SAP)大鼠肠道菌群结构、细菌移位随病程延长发生的改变。方法随机情况下将健康SD大鼠分为对照组(Control组n=10)、SAP组(n=30)。其中SAP组又分为6、12、24 h亚组(每亚组分别为10只大鼠)。按比例配置5%牛磺胆酸钠试剂,采用胰胆管逆行注射制作SAP模型。模型制备成功后分别于6、12、24 h后处死大鼠,检测血清淀粉酶、内毒素水平,观察胰腺及肠黏膜病理改变,用细菌培养法检测腹腔及脏器细菌移位率并用质谱仪鉴定培养出的阳性菌株,16S rDNA、实时荧光定量-聚合酶链式反应(real time-PCR)进行肠腔细菌定量分析。结果SAP组6、12、24 h亚组大鼠血清淀粉酶较对照组升高明显,且>3倍以上(U/L:2086±686 vs.8707±1672,9012±1505,8797±1298;P﹤0.01),胰腺组织及小肠黏膜出现明显病理损伤;随着病程延长,SAP组大鼠肠腔内大肠埃希菌不断增殖(×10^-5:4.53±1.26 vs.10.07±2.57,16.18±3.51,19.45±4.70;P﹤0.01),乳酸杆菌不断减少(×10^-3:23.12±4.46 vs.11.43±2.68,5.20±2.07.2.02±0.94;P﹤0.01),同样双歧杆菌也不断减少(×10^-5:23.30±6.00 vs.17.61±3.94,13.21±2.84,9.49±3.41;P﹤0.01);血清内毒素检测阳性率、肠系膜淋巴结及腹腔脏器细菌移位率均不断升高(血清内毒素:0 vs.30%,50%,60%;肝脏:0 vs.20%,50%,70%;胰腺:0 vs.30%,40%,60%;肠系膜淋巴结:0 vs.40%,70%,80%;P﹤0.01)。结论SAP大鼠肠道黏膜屏障受损,肠道菌群紊乱,肠腔内需氧菌与厌氧菌比例倒置,随着病程延长,以大肠埃希菌为主的条件致病菌不断增值,乳酸杆菌、双歧杆菌等厌氧益生菌不断减少,致使肠腔细菌及内毒素移位,其与SAP并发的胰腺及全身感染存在一定关系,且随着病程延长不断加重。

Objective This paper is to explore changes of intestinal flora and bacterial translocation in rats with severe acute pancreatitis(SAP).Methods The healthy SD rats were randomly divided into Control group(n=10)and SAP group(n=30).The SAP group was divided into 6,12 and 24 h subgroups,with 10 rats in each subgroup.The model of severe acute pancreatitis of rats was induced by injecting adversely 5%sodium taurocholate into the common billiary-pancreatic duct.The rats were sacrificed after 6,12 and 24 h respectively.The serum amylase and endotoxin level were detected and the pathological changes of the pancreas and intestinal mucosa were observed.Bacterial translocation rate in abdominal cavity and viscera was detected by bacterial culture,and positive colonies were identified by mass spectrometry:16SrDNA and real-time fluorescence quantitative polymerase chain reaction were used to quantitative analysis of enteric bacteria.Results The serum amylase(U/L)in the SAP group was significantly higher than that in the control group and more than three times(Control group=2086±686,6 h=8707±1672,12 h=9012±1505,24 h=8797±1298;P﹤0.01).The pancreatic tissue and the small intestinal mucosa showed obvious pathological damage.As the course of the disease prolonged,the intestinal Escherichia coli(×10^-5)in the SAP group continued to proliferate(Control group=4.53±1.26,6 h=10.07±2.57,12 h=16.18±3.51,24 h=19.45±4.70;P﹤0.01),the Iactobacillas(×10^-3)continues to decreased(Control group=23.12±4.46,6 h=11.43±2.68,12 h=5.20±2.07,24 h=2.02±0.94;P﹤0.01),the bifidobacterium(×10^-5)also continues to decrease(Control group=23.30±6.00,6 h=17.61±3.94,12 h=13.21±2.84,24 h=9.49±3.41;P﹤0.01)and the positive rate of serum endotoxin test,the mesenteric lymph node and the bacterial translocation rate of abdominal viscera increased(serun endotoxin:Control group=0,6 h=30%,12 h=50%,24 h=60%;liver:Control group=0,6 h=20%,12 h=50%,24 h=70%;pancreas:Control group=0,6 h=30%,12 h=40%,24h=60%;mesenteric lymph nodes:Control group=0,6 h=40%,12 h=70%,24 h=80%;P﹤0.01).Conclusion In rats with severe acute pancreatitis,the intestinal mucosal barrier is damaged,the intestinal flora is disturbed,the proportion of aerobic bacteria and anaerobic bacteria in the intestinal cavity is inverted.As the course of the disease is prolonged,the pathogenic bacteria of Escherichia coli are increasing,and the anaerobic probiotics,such as Lactobacillus and Bifidobacterium,are constantly decreasing,causing intestinal bacteria and endotoxin.There is a certain relationship between displacement and SAP and concurrent pancreas and systemic infection.