应用套式聚合酶链反应技术分析细菌与胆固醇结石形成关系的初步研究

A preliminary study of the relationship between the formation of cholesterol gallstones and bacteria by the nested primers polymerase chain reaction technique

目的 :旨在从分子水平研究细菌感染与胆固醇结石形成之间的关系 ,以期阐明胆固醇结石发病的分子机理和探讨可能防治的新途径。 方法 :从 36例胆汁细菌培养阴性的胆囊胆固醇结石中提取 DNA,应用套式聚合酶链反应 ( NP- PCR)技术 ,从中特异性地扩增细菌 DNA片段。 结果 :发现 30例 ( 83.33% )胆固醇结石中有细菌 DNA存在。其中胆固醇含量在 30 %～ 69%之间 6例 ( 6/ 6) ;70 %～ 90 %之间 14例 ( 14/ 17) ;>90 % 10例 ( 10 / 13) ,尚未发现结石中胆固醇和水分含量多少与细菌 DNA存在与否有关。采用 16S r RNA基因序列对照分析鉴定细菌种类 ,在 10例( 2 7.78% )胆石中发现与大肠杆菌相似的 DNA片段 ;从 8例 ( 2 2 .2 2 % )胆石中得到痤疮丙酸杆菌型 DNA序列 ;2例 ( 5.56% )胆石中残留的细菌 DNA片段与化脓性链球菌相关 ;8例 ( 2 2 .2 2 % )胆石内细菌的 DNA片段具有多样性 ,类似于大肠杆菌、痤疮丙酸杆菌、化脓性链球菌、艰难梭状芽胞杆菌或其它未鉴定细菌 ,可能有多种细菌混合感染。另有 2例胆石的细菌 DNA分子量较低 ,不同于大肠菌、痤疮丙酸杆菌、化脓性链球菌、金黄色葡萄球菌和铜绿色假单胞菌 ,归类于其它未鉴定细菌。 结论 :证实多数胆固醇结石内有细菌 DNA存在。至于这些微生物在胆固醇结石形?

Objectives:To study the relationship between the formation of cholesterol gallstones and bacterial infections with methods of molecular biology,expound the choles- terol gallstone molecular pathogenesis,and search for a possible new way of preventionand treatment. Methods:Bacterial gene fragments were amplified by NP- PCR in vitro from DNA extracted from cholesterol gallbladder gallstones with negative bacterial culture.Com- parative1 6 S ribosomal RNA sequence analysis was used for elucidation of bacterial identifi- cation. Results:The gallbladder gallstones of36 patients were analysed.Bacterial DNA was found in the stones of30 patients(83.33% ) ,among which gallstones with cholesterol contentbetween30 % to6 9% in6 patients(6 /6 ) ,70 % to90 % in1 4patients(1 4/1 7) ,and more than 90 % in 1 0 patients (1 0 /1 3) .There was no difference either in cholesterol and wa- ter content or in harboring bacterial DNA of gallstones.E.coli- related DNA fragments were found in the stones of1 0 patients(2 7.78% ) .Propionibacteria type DNA was found in the stones of 8patients(2 2 .2 2 % ) .Stones of 2 patients(5 . 5 6 % ) harbored bacterial gene frag- ments with similarity with Streptococcus pyogenes.A more heterogenous sequence collection was found in 8patients(2 2 .2 2 % ) and could be assigned to the mixed infections including E. coli,Propionibacteria and Streptococcus pyogenes or some other unidentified bacteria such as Clostridium diffecile.Another stones of2 patients(5 .5 6 % ) had bacterial DNA with lower molecular weight,different from E. coli,Propionibacteria,Streptococcus pyogenes,Staph. aureus and Pseudomonas aeruginosa,and might be related to some unidentified bacteria. Conclusions:Most cholesterol gallstones harbor bacterial DNA.Further studies remains to be done before it can be decided whether these microorganisms are innocentbystanders or ac- tive participants in cholesterol gallstone formation.