【作者】 朱雷明；

【导师】 蔡端；

【作者基本信息】 复旦大学 ， 外科学， 2003， 博士

【摘要】 胆固醇类结石是严重影响人民身体健康的常见病和多发病。但是胆固醇类结石的形成机制尚未完全阐明。其中与胆汁成分的代谢变化、胆囊运动失调的关系等有了较多的研究，但未考虑细菌感染的因素。近年来在胆固醇类结石中发现有丰富的细菌DNA，提示细菌感染与胆固醇类结石的形成可能有关。 本研究运用分子生物学方法，首次检测了胆固醇结石病人的胆石、胆汁和胆囊粘膜中的细菌DNA，探讨其与胆汁和胆囊粘膜中免疫球蛋白含量的相关性，并与非胆石症病人进行对照。首次观察了胆固醇类结石中检测到的细菌对胆汁中胆固醇晶体形成的影响以筛选致石细菌，并研究其对胆汁热力学和动力学的影响，探讨其促成核活性的来源和性质。 第一部分 胆固醇类结石与非胆石症病人胆道细菌感染状况及与免疫球蛋白相关性的对比研究 目的：胆固醇类结石中细菌DNA的检出率远高于细菌培养的阳性率。为明确细菌在胆固醇结石发病机理中的地位，比较胆固醇类结石病人和非胆石症病人胆道细菌感染情况以及与免疫球蛋白的相关性。方法：用不依赖细菌培养的半定量PCR和16SrRNA序列对照法，检测38例胆固醇类结石病人胆囊粘膜、胆汁和胆石中细菌DNA，与17例非胆石病人对照。同时用散射比浊法测粘膜和胆汁中IgA、IgG、IgM含量。结果：(1)胆石组和非胆石组胆汁细菌DNA阳性率分别为77％和67％，菌落数分别为3.23cfu(对数值)和2.43cfu，胆囊粘膜细菌DNA阳性率分别为64％和69％，菌落数分别为3.28cfu和3.67cfu，两组间均无显著差异(P＞0.05)。(2)两组间胆汁和粘膜IgA、IgG、IgM含量也无显著差异(P＞0.05)。菌落数与免疫球蛋白含量不相关。(3)结石核心和外周的细菌DNA阳性率分别为79％和82％，菌落数分别为3.19cfu和3.26cfu；纯胆固醇结石细菌DNA阳性率为3／3例。结石中胆固醇含量与细菌DNA阳性率不相关。(4)胆石组细菌DNA阳性者与阴性者的胆囊粘膜IgA、IgG含量均有显著差异(P<0.05).(5)术前用抗生素者胆汁菌落数显著减少伊<0 .05)，但是用和未用抗生素者的胆石核心、外周或粘膜菌落数无显著差异(P>0.05)。(6)胆石组细菌种类主要为大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、不动杆菌、链球菌、鞘氨醇单胞菌、脆弱类杆菌和痊疮丙酸杆菌，胆石组菌种比非胆石组丰富，非胆石组菌种基本上均能在胆石组中找到。结论:(l)胆固醇结石与非胆石症病人胆道细菌感染率相似，胆固醇结石中存在细菌不足以证明细菌参与胆固醇结石的形成。(2)细菌可能引起胆囊粘膜免疫球蛋白的分泌，间接参与细菌结石形成。第二部分细苗对胆汁胆固醉晶体形成的形晌目的:观察不同细菌对胆汁胆固醇晶体形成的影响以筛选其中的致石细菌。方法: (I)观察大肠杆菌、铜绿假单胞菌、粪肠球菌、金黄色葡萄球菌、不动杆菌、无乳链球菌、鞘氨醇单胞菌、嗜麦芽窄食单胞菌、脆弱类杆菌、产抱梭菌、艰难梭菌和座疮丙酸杆菌在未离心人胆汁、离心人胆汁和模拟胆汁中对胆汁胆固醇晶体成核时间(NT)以及对胆汁中的起始胆固醇晶体量和最大胆固醇晶体量的影响，筛选可能的致石细菌。(2)在含致石细菌的模拟胆汁中加入10m加卜一80mM的游离胆红素(UCB)，观察胆汁NT的变化。结果:(l)以加入生理盐水的胆汁为对照，痊疮丙酸杆菌在三种胆汁体系中无缩短NT的作用，提示无促成核活性。 (2)其他受检细菌使未离心的人胆汁NT比加入生理盐水的对照胆汁明显缩短，胆汁中形成的最大晶体量明显高于对照组伊劝.05);但在离心的人胆汁中无此差异 (P>0.05)。(3)在模拟胆汁中仅铜绿假单胞菌和粪肠球菌显示降低N护I，的活性，细菌浓度1护cfi刀inl至1015c几lm】间NT无差异(P>0.05)，但是细菌浓度越高，晶体量越大(P<0.05)。(4) ucB能降低模拟胆汁NT(p<0 .05)，但UcB浓度1 OniM‘80血M间NT的改变与浓度无关(P>0.05)，UCB与铜绿假单胞菌或粪肠球菌无协同促成核效应。结论:(l)某些细菌物质可能与人胆汁中促成核因子有协同作用。(2)铜绿假单胞菌和粪肠球菌能直接降低胆汁NT，具有促成核作用;而座疮丙酸杆菌不能改变胆汁NT，其致石机理可能与铜绿假单胞菌和粪肠球菌不尽相同。(3)UCB可降低模拟胆汁的NT，但是与细菌无协同作用。国的：探讨铜绿假单胞菌、粪肠球菌和大肠杆菌对胆汁热力学平衡的影响。方法：在模拟胆汁中加入铜绿假单胞菌、粪肠球菌和大肠杆菌，观察模拟胆汁的形态变化，记录胆汁中粘附物质析出时间…nset ime of adhesion），用粘度计测量胆汁的粘度变化，同时测定胆固醇、卵磷脂、胆汁酸盐浓度变化。结果：＊）铜绿假单胞菌和粪肠球菌使模拟胆汁粘度增加，胆汁中溶质析出并粘附在试管壁上，胆固醇溶解度下降，与未加入细菌的模拟胆汁比较有显著差异则．05卜 ＊）加入大肠杆菌的模拟胆汁与未加入细菌的模拟胆汁比较，胆汁粘度和胆汁中胆固醇溶解度无显著差异脚．05），无胆汁成分析出。结论：铜绿假单胞菌和粪肠球菌使模拟胆汁粘度增加，胆汁中溶质析出、粘附，胆固醇溶解度下降，可能促进胆固醇结石的形成。第四部分用组团配用、勇同阳和朋m一位的输协用燃的研究目?更多还原

【Abstract】 Cholesterol gallstone disease remains a common cause of serious morbidity and mortality.The natural history of cholesterol gallstone pathogenesis is not thoroughly understood Many studies suggested that cholesterol gallstone formation related to the disorder of bile metabolism and gallbladder contractility. But little attention has been payed to bacteria. Recently rich amount of bacteria DNA had been found in most cholesterol gallstones, which indicates a possible link between bacteria and cholesterol gallstone formation.Our study is the first one to detect and quantify the bacteria DNA from gallstone,gallbladder mucosa, gallbladder bile in cholesterol gallstone patients and from gallbladder mucosa, gallbladder bile in non-gallstone patients, coupled with the correlation with immunoglobulins concentration in bile and gallbladder mucosa. We are also the first one to observe the influence of bacteria found in cholesterol gallstones on the cholesterol nucleation time(NT) to find out the lithogenic ones, and study their influence on the thermodynamic balance of bile system, explore the source and mechanism of their pronucleation activity.The first part The comparison of bacterial infection state between cholesterol gallstone patients and non-gallstone individues, and the correlation with immunoglobulins concentration in bile and gallbladder mucosa[Aim]: The frequence of bacteria DNA detected with PCR in cholesterol gallstones is much higher than the culture results. To evaluate the status of bacteria in cholesterol gallstone pathogenesis, we compare the bacterial infection state between cholesterol gallstone patients and non-gallstone individues, coupled with the correlation with immunoglobulins concentration in bile and gallbladder mucosa. [Methods): Culture independent, half-quantitative PCR and comparative 16S rRNA analysis were used to detect bacteria from gallstones, bile and gallbladder mucosa in 38 cholesterol gallstone patients and 17 non-gallstone individuals. IgA, IgM, IgG concentrations of bile andgallbladder mucosa were also measured parallelly by the method of nephelometry. [Results]: 1. The positive rate of bacteria DNA in bile of gallstone patients and controls was 77% and 67%, with concentrations of 3.23 cfu(log value) and 2.43 cfu respectively. The positive rate of bacteria DNA in mucosa was 64% and 69%,with concentrations of 3.28cfu and 3.67 cfu respectively. No significant difference existed between two groups, including the concentrations of immunoglobulin(P>0.05). No positive correlation was observed between immunoglobulin and bacteria concentrations. 2. The positive rate of bacteria in core and periphery part of gallstones was 79% and 82%, with bacteria concentrations of 3.19 cfu and 3.26 cfu respectively. All three pure cholesterol stones harbored trace of bacterial DNA. No positive correlation existed between the incidence of bacteria and the percentage of cholesterol in the stone. 3. There was a sharp distinction for mucosa IgA, IgG concentrations between mucosa bacteria positive and negative ones in gallstone patients(P<0.05).4. The bile bacteria concentrations fell significantly after accepting a dosage of antibiotics right before operation, while no change on core or periphery part of gallstone and mucosa was observed. 5. The bacteria sequences in cholesterol gallstone group included Propionibacterium acne, E.coli, Pseudomonas aeruginosa, Streptococcus, Acinetobacter, Sphingmonas, Bacteroides fragilis. Cholesterol gallstone group had more bacteria sequences than non-gallstone group, most of sequences in latter group could be found in cholesterol gallstone group. [Conclusions]: 1 .There is a similar biliary infection rate between gallstone patients and controls. Bacteria found in biliary tract is not sufficient to draw a definite link to cholesterol gallstone formation. 2. However, The cholesterol group has more bacteria sequences than the non-gallstone group, some of which overlapped in two groups. 3.The bacteria on mucosa maybe indirectly lithogenic through IgAJgG content increa更多还原