2型糖尿病合并急性胰腺炎大鼠肠道微生物ERIC-PCR指纹图谱的分析

Analysis of the microorganism ERIC-PCR fingerprint in rats suffering type 2 diabetes mellitus combined with acute pancreas injury

目的应用肠杆菌基因间重复共有序列-聚合酶链式反应(enterobacteria repetitive intergenic consen sussequencespolymerase chain reaction,ERIC-PCR)技术检测2型糖尿病合并急性胰腺炎后大鼠肠道内菌群的数量及多样性的改变情况。方法40只10周龄雄性大鼠,其中20只Wistar大鼠,随机分为空白对照组(QB组)、单纯急性胰腺炎大鼠模型(AP组);20只Goto-kakisaki自发性2型糖尿病大鼠(GK大鼠)随机分为2型糖尿病组(DM组)、2型糖尿病合并急性胰腺炎组(DAP组);每组10只,分别标号。其中AP组、DAP组作为造模组。检测DAP组和AP组24 h血清胰淀粉酶活性水平和随机血糖。48 h后:(1)处死各组大鼠,取胰腺观察病理损伤程度并以改良Schmidt法为标准进行胰腺组织病理评分。(2)采集各组大鼠新鲜粪便,并提取新鲜大鼠粪便基因组,进行基因组扩增,将PCR产物点样于琼脂糖凝胶,制作目的 ERIC-PCR指纹图谱;(3)同时将采集的新鲜粪便处理后制成不同浓度梯度细菌悬浊液,接种于BBL琼脂培养基、MRS肉汤培养基、SS琼脂培养基、MAC琼脂培养基上培养并计数。结果 (1)AP组、DAP组病死率分别是20%(2/10)和40%(4/10),QB组、DM组无死亡,DAP组病理评分最高;(2)DAP组术后24 h血糖水平较AP组升高,差异具有统计学意义(t=2.24,P<0.05);DAP组术后24 h血淀粉酶较AP组无明显变化,两组比较差异无统计学意义(t=6.40,P=0.53);(3)肠道菌群多样性指数、ERIC-PCR指纹图谱及相似性聚类分析显示,DAP组特征性条带数量减少且肠道菌群多样性改变;(4)4组间肠道微生物菌群比较,差异具有统计学意义。进一步两两比较,DAP组的双歧杆菌和乳酸杆菌低于QB组和AP组,差异具有统计学意义(P<0.05);DAP组的大肠杆菌和变形杆菌高于QB组和AP组,差异具有统计学意义(P<0.05)。结论 (1)2型糖尿病大鼠并发急性胰腺炎后肠道菌群的结构均发生改变,菌群多样性减少;益生菌减少及条件致病菌增多;(2)肠道菌群的变化参与2型糖尿病合并急性胰腺炎的病程进展。

Objective The objective of this study was to use the enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction （enterobacteria repetitive intergenic consen sussequences-polymerase chain reaction, ERIC-PCR） technologyto detect the volume and diversity of intestinal flora in rats suffering type 2 diabetes mellitus and acute pancreatitis. Methods 40 malerats aged 10 weeks were divided into two groups, of which 20 Wistar rats were randomly divided into blank control group （QB groupand simple acute pancreatitis rats model （AP group）; 20 Goto-kakisaki spontaneous type 2 diabetic rats （GK rats） were randomly dividedinto type 2 diabetes group （DM group） and type 2 diabetes mellitus and acute pancreatitis group （DAP group）. Each group included 10rats and was given labels respectively. The AP group and DAP group were used as modules and their serum levels of pancreatic amylaseactivity and random blood glucose were detected at the time point of 24 h. After 48 h： （1） The rats were executed and their pancreas weretaken for examination of the pathological damage severity. And the pancreatic tissue was pathologically graded by the modified Schmidmethod. （2） Fresh feces of rats were collected and its genome were extracted, carrying out genomic amplification. ERIC-PCR fingerprintswere produced by doting the product of PCR on the agarose gel. （3） The fresh feces were processed into bacterial suspensions on differenconcentration gradient, vaccinated in the BBL AGAR media, MRS broth, SS AGAR medium, MAC to cultivate on the AGAR medium and were counted. Results （1） The fatality rate of AP group and DAP group was 20% （2/10） and 40% （4/10） respectively while there was no death in the QB group and DM group, and the pathological score of DAP group was the highest. （2） The blood glucose in DAP group at the time point of 24 h following the operation was higher than that in AP group, and the difference was statistically significant （t=2.24, P〈0.05）. The blood amylase in the DAP group at the time point of 24 h following the operation barely changed as compared to the AP group, and the difference was not statistically significant （t=6.40, P=0.53）. （3） The intestinal flora diversity index, ERIC-PCR fingerprint and similarity cluster analysis showed that the volume of characteristic bands of DAP group decreased and the diversity of intestinal flora changed. （4） The differences of intestinal microbial flora between the four groups were not statistically significant. Further comparison showed that the bifidobacterium and lactobacillus of the DAP group were lower than those of QB group and AP group, and the differences were statistically significant （P〈0.05）. The escherichia coli and the modified bacillus in the DAP group was higher than those in the QB group and AP group （P〈0.05）. Conclusions （1） The structure of intestinal flora in rats changes after suffering acute pancreatitis combined with type 2 diabetes mellitus, while its diversity and probiotics decrease and opportunistic pathogens increase; （2） the progression of type 2 diabetes mellitus combined with acute pancreatitis are accompanied with changes in intestinal flora.