应用16S rRNA PCR技术评价益生菌VSL#3对实验性结肠炎大鼠肠道菌群的调节作用

Application of 16S rRNA-Based Polymerase Chain Reaction in the Evaluation of the Probiotics Treatment(VSL#3)on TNBS-Induced Colitis Through Modulation of Gut Flora in Rats

目的:应用细菌的16S rRNA/rDNA序列设计引物,PCR检测经益生菌治疗后肠道菌群的相对变化,观察益生菌VSL#3对大鼠实验性结肠炎的保护作用。方法:将24只SD大鼠以三硝基苯磺酸(TNBS)乙醇溶液一次性灌肠造成急性实验性结肠炎模型,并随机分成3组,即模型组、VSL#3组和5-氨基水杨酸(5-ASA)组,分别给予生理盐水、益生菌VSL#3、5-ASA灌胃。观察各组大鼠一般情况、体重、大便的改变。1周后处死大鼠,观察结肠病理变化,比较每组炎症积分。留取大便样本,分别进行双歧杆菌、大肠杆菌、乳酸杆菌的培养、计数。应用细菌的16S rRNA/rDNA序列设计引物,对大鼠肠道双歧杆菌、大肠杆菌、乳酸杆菌和通用细菌的16S rDNA基因进行PCR检测,并计算前3种细菌16S rDNA基因的相对扩增产物以观察肠道菌群的变化。结果:TNBS造模1周内各组大鼠均出现懒动、食量下降、腹部膨胀、毛色无光泽、体重下降、腹泻、便血的表现。处死后发现每组均有不同程度的结肠、回盲部扩张,局部结肠淤血、与周围组织黏连,肠黏膜有出血点、溃疡甚至糜烂或肠黏膜脱落,以模型组最明显。VSL#3组和5-ASA组的炎症评分显著低于模型组(P<0.05)。经PCR及细菌培养的方法均显示VSL#3组肠道大肠杆菌含量低于另两组(均为P<0.01),而双歧杆菌和乳酸杆菌含量显著高于另两组(P<0.01或P<0.05)。结论:利用细菌16S rRNA/rDNA设计引物,对多种肠道细菌相对含量进行PCR检测,发现益生菌VSL#3可通过调节肠道菌群,达到对大鼠实验性结肠炎的治疗效果。

Objective：To investigate the effect of probiotics VSL#3 on the gut bacteria of TNBS-induced colitis rat models by PCR using 16S rDNA primers,and to determine whether VSL#3 has anti-colitis effects through modulating gut flora or not.Methods：Trinitro-benzene-sulfonic acid（TNBS）induced colitis models were established in 24Sprague-Dawley rats.The rats were randomly divided into three groups as model group,VSL#3 group,and 5-aminosalicylic acid（5-ASA）group according to the treatment.Rats＇behaviors,body weight,and feces changes were recorded.Seven days after modeling,all rats were sacrificed,colon and feces samples were collected for HE staining and gut flora determination.PCR using 16S rDNA primers was adopted to assay the relative amount of bacterial 16S rDNA gene PCR products,and the results were compared with those of bacterial counting by conventional culturing methods.Results：All rats showed colitis features such as body loss,abdominal distension,diarrhea,and blood stool.Pathologic changes were severer in model group than in VSL#3 and 5-ASA groups,which includes inflammatory cell infiltration,destruction of crypt structures,and ulcer or erosion of the mucosa.Both PCR and bacterial counting results showed that both Lactobacillus and Bifidobacteria population were higher,while E.coli strains were less in VSL#3 group than in the other groups.Conclusion：VSL#3 could alleviate the TNBS-induce colitis through balancing the gut flora of TNBS-induced colitis rats.PCR using 16S rDNA primers can be adopted to determine the amount of gut bacteria accurately and specifically.