实时荧光定量PCR法研究结直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化

Real-time fluorescence quantitative-PCR analysis of the variation of Bacteroides,Fusobacterium and Clostridium in patients with colorectal cancer

目的通过研究结、直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化,揭示肠道相关菌群改变在大肠癌发病中的作用及意义。方法收集术前结、直肠癌患者粪便标本40例及正常对照标本40例,根据细菌的靶基因序列设计特异性引物。提取待测粪便标本细菌DNA,应用SYBR Green I实时荧光定量PCR测定不同细菌的数量。结果正常对照组与实验组粪便中细菌数量分别为拟杆菌属(8.76±0.77;9.85±0.88)、梭杆菌属(7.94±1.25;10.0±1.65)、梭菌属(3.54±0.70;6.56±0.68),拟杆菌属中的脆弱拟杆菌为(2.12±0.48;4.07±1.77)、梭杆菌属中的坏死梭杆菌为(2.31±0.26;7.62±2.68)及梭菌属中的肉毒梭菌为(2.76±1.16;5.43±1.21),实验组数量均明显增多(P<0.05)。结论结、直肠癌患者粪便中拟杆菌属、梭杆菌属和梭菌属的数量较正常对照明显增多,提示结、直肠癌的发生发展与肠道菌群有明显关系。

Objective To analyze changes in the mumber of Bacteroides, Fusobacterium and Clostridium in color- ectal cancer patients by real-time fluorescence quantitative PCR and reveal the role and significance of gut microflora in colorectal cancer-associated molecular pathogenesis. Method The feces of preoperative colorectal cancer patients （ n = 40） and healthy volunteers （ n = 40） were collected. Specif primers were designed according to the target gene sequences of Bac- teroides ,Fusobacterium and Clostridium. Bacterial genome DNA extracted from fecal samples were quantified by SYBR Green I real-time fluorescence quantitative PCR to analyze bacterial amounts. Result In normal control group and study group the levels of Bacteroides （8.76 ± 0.77 ; 9.85 ± 0.88）, Fusobacterium （7.94 ± 1.25 ; 10.0 ± 1.65 ） and Clostridium （3.54 ± 0.70 ; 6.56± 0.68） ,B. fragilis （2.12 ±0.48 ; 4.07 ±1.77 ）, F. necrosis （ 2.31 ± 0.26 ; 7.62 ± 2.68） and C. botulinum （2.76 ±1.16 ; 5.43 ± 1.21 ） were significantly higher than those of healthy controls （ P 〈 0.05 ）. Conclusion The fecal Bacteroides, Fusobacterium and Clostridium composition in colorectal cancer patients are significantly higher than those of healthy controls, which suggests that the colorectal cancer is related to intestinal microflora.