胆囊结石病患者胆道系统的螺杆菌DNA

Detection of Helicobacter sp. in the biliary system of patients with cholelithiasis

目的 近来 ,螺杆菌属细菌与胆道疾病的关系日益受到重视 ,并在胆固醇结石中检测到细菌DNA。为进一步研究胆囊结石病与螺杆菌感染的关系 ,对胆囊结石患者的胆石、胆汁 ,胆囊黏膜行螺杆菌 16SrRNA基因检测。方法 研究对象为 2 9例胆囊结石病患者。胆汁和胆囊黏膜标本接种于需氧、厌氧和非选择性微需氧培养基培养。利用两对特异性的螺杆菌 16SrRNA基因引物对患者的胆石、胆汁、胆囊黏膜标本行nested PCR扩增 ,利用特异的螺杆菌DNA探针 ,对PCR产物行Southern杂交检测 ,并对 1例胆囊黏膜PCR产物直接测序 ,将测序结果与基因库中的标准螺杆菌株序列作比较 ,采用Neighbor joining法绘制种系发生树。氧化酶法测定胆石胆固醇含量。 结果 胆汁和胆囊黏膜标本经需氧、厌氧、微需氧培养 ,未发现螺杆菌生长。 2 9例胆石标本nested PCR分析 ,2例阳性 (6 .9% ) ,2 3例胆汁中 9例阳性 (39.1% ) ,2 9例胆囊黏膜中 2 2例阳性 (75 .9% )。PCR阳性的标本Southern杂交均为阳性。 1例胆囊黏膜标本的PCR扩增产物纯化后直接测序 ,证实为螺杆菌属细菌。 2 9例胆囊结石的平均胆固醇含量为 (6 2 .2± 3.8) % ,PCR结果阳性的 2例胆石为胆固醇为主的混合性结石。结论 螺杆菌DNA存在于胆囊结石、胆汁及胆囊黏膜中 。

Objective Recently, studies have been focusing on the relationship of Helicobacter sp. and biliary disease. Bacterium DNA has been found in cholesterol stone. To study the relationship between cholelithiasis and the infection of Helicobacer sp., we used nested primers polymerase chain reaction (nested-PCR) to amplify Helicobacter sp.16S rRNA gene fragments in gallstones, bile and gallbladder mucosa. Methods All samples were obtained from 29 patients with cholelithiasis. Aerobic, anaerobic, and microaerophilic cultures were performed for bile and gallbladder mucosa. 16S rRNA gene fragments of Helicobacter sp. were amplified by nested-PCR with two couples of primers. Southern blot hybridization was performed with a specific Helicobacter probe. PCR product from gallbladder mucosa in one patient was sequenced directly and subjected to phylogenetic analysis. Cholesterol concentration in gallstones was analyzed. Results The results of Helicobacter culture were negative. However, 2 of 29 gallstones (6.9%), 9 of 23 bile (39.1%) and 22 of 29 gallbladder tissues (75.9%) were positive for Helicobacter sp. by nested-PCR analysis. Each sample positive by PCR was also positive by Southern hybridization. PCR product from gallbladder mucosa was confirmed as Helicobacter sp. clustering with Helicobacter rodentium-like through phylogenetic analysis. The average cholesterol concentration in gallstones was (62.2±3.8)% and both gallstones positive for Helicobacter sp. by PCR had more than 50% cholesterol. Conclusions It was suggested that Helicobacter sp. is present in some gallstones, bile and gallbladder mucosa from patients with cholelithiasis. Infection of Helicobacter sp. in the biliary system may be one of the risk factors for gallstone formation.