细菌在胆固醇结石形成中作用的研究
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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?
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 and
gallbladder 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
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?
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 and
gallbladder 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
引文
1． 黄志强主编．当代胆道外科学．上海科技文献出版社，1998，249-260．
2． Maki T. Pathogenesis of calcium bilirubinate gallstone: role of E. coli, beta-glucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation. Ann Surg 1966,164: 90-100．
3． Strasberg SM,Clavien PA, Harvey PR. Pathogenesis of cholesterol gallstones. HPB Surg 1991,3:79-102
4． Hatsushika S, Tazuma S, Kajiyama G. Nucleation time and fatty acid composition of lecithin in human gallbladder bile. Scand J Gastroenterol 1993,28:131-136．
5． Moser AJ, Abedin MZ, Roslyn JJ. Increased biliary protein precedes gallstone formation. Dig Dis Sci 1994,39:1313-1320．
6． Luk As, Kaler EW,Lee SP. Protein lipid interaction in bile: effects of biliary proteins on the stability of cholesterol-lecithin vesicles. Biochim Biophys Acta 1998,1390: 282-292．
7． Mansour A, Dawoud I, Gad-El-Hak N. The potential site of disordered gallbladder contractility during the early stage of cholesterol gallstone formation. Hepatogastroenterology 1998,45:1404-1409．
8． Truedson H, Elmros T, Holm S. The incidence of bacteria in gallbladder bile at acute and elective cholecystectomy. Acta Chir Scand 1983,149:307-313．
9． Moore EW. Biliary calcium and gallstone. Hepatology 1990,12:206s-214s.
10 Kaufman HS, Magnuson TH, Pitt HA, et al. The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown pigment gallstones. Hepatology 1994,19: 1124-1132．
11 Okido M, Shimizu S, Ostrow JD, et al. Isolation of a calcium-regulatory protein from black pigment gallstones: similarity with a protein from cholesterol gallstones. Hepatology 1992,15: 1079-1085．
12 Swidsinsky A, Ludwig W, Pahlig H, et al. Molecular genetic evidence of bacterial colonization of cholesterol gallstones. Gastroenterology 1995,108: 860-864．
13 Weisberg WG, Barns SM, Pelletier DA, et al. 16S ribosomal DNA amplification for phylogenetic study. 1991,173: 697-703．
14 Greisen K, Loeffelholz M, Purohit A, et al. PCR primes and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clinic Microbiology 1994,32: 335-351．
15 Lee DK, Tarr P, Haigh WG, et al. Bacterial DNA in mixed cholesterol gallstones. Am J Gastroenterol 1999,94:3502-3506．
16伍晓汀，肖路加，黎介寿，等．由胆固醇结石提取DNA进行套式聚合酶链反应的研究．中华外科杂志1997，35：663-666．
17 Soloway RD, Crowther RS. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis. Gastroenterology. 1995,108: 934-936．
18 Swidsinski A, Lee SP. The role of bacteria in gallstone pathogenesis. Front Biosci 2001,6:E93-E103．
19 Cariati A, Cetta F. Re: Kawai et al.-Bacteria are not important in the formation of pure cholesterol stones. Am J Gastroenterol 2002,97:2921-2923．
20 Swidsinski A, Khilkin M, Pahlig H, et al. Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstone. Hepatology 1998,27:662-665．
21 Fromm H, Amin P, Klein H, et al. Use of a simple enzymatic assay for cholesterol analysis in human bile. J Lipid Res 1980,21:259-261．
22 Gurantz D, Laker MF, Hoffmann AF. Enzymatic measurement of choline-containing phospholipids in bile. J Lipid Res 1981,22:373-376．
23 Turley SD, Dietschy JM. Re-evaluation of the 3 a-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978; 19:924-928．
24 Carey MC. Critical tables and cholesterol saturation of bile. J Lipid Res 1978,19:945-955．
25 Ohdan H, Oshiro H, Yamamoto Y, et al. Bacteriological investigation of bile in patients with cholelithiasis. Surgery Today 1993,23:390-395．
26 Fox JG, Dewhirst FE, Shen Z, et al. Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology, 1998,114:755-763．
27 Figura N, Cetta F, Angelico M, et al. Most Helicobacter pylori-infected patients have specific antibodies, and some also have H.pylori antigens and genomic material in
bile. Is it a risk factor forgallstone formation? Dig Dis Sci, 1998, 43: 854-862.
28 Kawai M, Iwahashi M, Uchiyama K, et al. Gram-positive cocci are associated with the formation of completely pure cholesterol stones. Am J Gastroenterol 2002, 97:83-88.
29 Prouty AM, Schwesinger WH, Gunn JS. Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp. Infect Immun, 2002, 70: 2640-2649.
30 韩文胜，石景森，卓健生，等．检测胆囊结石胆汁中L型细菌的研究及其意义．中华实验外科杂志2002，18：207-208．
31 吕厚东，曹卉，山长武，等．人及多种动物胆汁诱导出细菌L型的实验研究．济宁医学院学报1995，18：22-23．
32 Goodhart GL, Levison ME, Trotman BW, et al. Pigment vs cholesterol cholelithiasis.Bacteriology of gallbladder stones, bile, and tissue correlated with biliary lipid analysis. Am J Dig Dis 1978; 23:877-882.
33 黄美雄，朱明炜，宋京海，等．胆道感染细菌的变迁及细菌对抗生素敏感性分析．中华肝胆外科杂志2001，7：272-274．
34 朱雷明，陈为欢，蔡端，等．T管胆汁细菌培养及对抗生素敏感性分析．外科理论与实践经验2003，8：124-126．
35 Wetter LA, Hamadeh RM, Griffiss JMCL, et al. Difference in outer membrane characteristics between gallstone associated bacteria and normal bacteria flora. Lancet 1994,343:444-447.
36 Monstein HJ, Jonsson Y, Zdolsek J, et al. Identification of Helicobacter pylori DNA in human cholesterol gallstone. Scand J Gastroenterol 2002,37:112-119.
37 Myung SJ, Kim MH, Shim KN, et al. Detection of Helicobacter pylori DNA in human biliary tree and its association with hepatolithiasis. Dig Dis Sci 2000,45:1405-1412.
38 La Scola B, Barrassi L, Raoult D. Isolation of new fastidious alpha proteohacteria and Afipia felis from hospital water supphes by direct plating and amoebal co-culture procedures. FEMS Microbiol Ecol 2000.34:129-137.
39 Harvey PR, Upadhya GA, Strasberg SM,. Immunoglobulins as nucleation proteins in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 1991,266:13996-14003．
40 Abei M, Schwarzendrube J, Nuutinnen H, et al. Cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, a 1-acid glycoprotein, phospholipase C,and aminopeptidase N. J Lipid Res 1993,34:1141-1148．
41 Miquel JF, Nunez L, Rigotti A, et al. Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles. FEBS Lett 1993,318:45-49．
42 de Brujin MAC, Mok KS, Out T, et al. Immunoglobulins and a 1-acid glycoprotein do not contribute to the cholesterol crystallization-promoting effect of Concanavalin A-binding biliary protein. Hepatology 1994,20: 626-632．
43 Alissa K, Saunier P, Russo M, et al. Neonatal cholestatic lithiasis associated with E. coli infection. Arch pediatr 1996,3: 144-146．
44 Kibe A, Dudley MA, Halpem DZ, et al. Factors influencing cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile. J Lipid Res 1985, 26:1102-1111．
45 Holan KR, Holzbach RT, Hermann RE, et al. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979, 77:611-617．
46 de 1a porte PL, Lafont H, Domingo N, et al. Composition and immunofluorescence studies of biliary "sludge" in patients with cholesterol or mixed gallstones. J Hepatol 2000,33:352-360．
47王巍，韩天权，张圣道．胆固醇结石病胆汁游离脂肪酸的水平及其临床意义．中华外科杂志1995，33：659-661．
48 Halpem Z, Goldman G, Peled Y, et al. Free fatty acids have nucleating effects in model biles. Liver 1992,12:107-111．
49 Kabongo Muamba ML. Exoenzymes of Propionibacterium acnes. Can J Microbio 1982,28:758-761．
50 Nakamara T, Taniguchi H, Takeuchi K, et al. Phospholipase C of an oral strain of Propionibacterium. Purification and partial characterization. Zbl Bakt Hyg A 1984,257:20-26．
51 Pattinson NR, Willis KE. Effect of phospholipase C on cholesterol solubilization in model bile. Gastroenterology 1991, 101: 1339-1344.
52 Lichtenberg D, Ragimova S, Peled Y, et al. Phospholipid peroxidation as a factor in gallstone pathogenesis. FEBS Lett 1988,228:179-181.
53 马保金，蔡端，张延龄，等．胆汁泡蛋白成核活性及其氨基酸序列分析的研究．外科理论与实践1999，4：22-23．
54 项建斌，蔡端，张延龄，等．33.5kDa胆汁泡蛋白促成核活性及其对泡形态学影响的研究．肝胆胰外科杂志1999，1：8-10．
55 Miguel JF, von Ritter C, Del Pozo R, et al. Fibronectin in human gallbladder:cholesterol pronucleating and/or "link" protein? Am J Physiol 1994, 267: G393-400.
56 焦宛，张延龄，蔡端，等．胆汁促成核糖蛋白的分离提纯及理化性质研究．中华外科杂志1994，32：271-272．
57 励峰，蔡端，莫汉庆，等．ELISA法检测200kDa糖蛋白．中华消化杂志1997，17：333-335．
58 Miquel JF, Rigotti A, Rojas E, et al. Isolation and purifiaction of human biliary vesicles with potent cholesterol-nucleation-promoting activity. Clin Sci 1992, 82:175-180.
59 Miquel JF, Nunez L, Amigol L, et al. Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile.Gastroenterology 1998,114: 1016-1023.
60 项建斌，蔡端，张延龄，等．胆汁泡蛋白促成核活性以及在不同人群含量的分布．中华实验外科杂志2001，1：83．
61 Ostrow JD, Celic L, Mukerjee P, et al. Molecular and micellar associations in the pH-dependent stable and metastable dissolution ofunconjugated bilirubin by bile salts. J Lipid Res 1988,29: 335-348.
62 Fu XP, Zhou XS, Zhang WH, et al. "Bilirubin-calcium compound" precipitation and the effect ofbile salts on it;the pathogenesis of pigment gallstone. Chinese Med J 1998,728-738.
63 Leung JW, Liu YL, Leung PS, et al. Expression of bacterial beta-glucuronidase in human bile: an in vitro study. Gastrointest Endose 2001,54:346-350.
64 蔡端，张延龄，邬建国，等．胆汁中胆固醇和胆红素含量的相关性研究．上海医学1989，12：709-711．
65 Kaufman HS, Magnuson TH, Webb TH, et al. Bilirubin monoglucuromde promotes cholesterol gallstone formation. J Surg Res 1991,50:504-509.
66 Zhu XG, Tu XQ, Wang RH, et al. Observation on solubility and pro-nucleative activity of monoconjugated bilirubin. Chinese Med J 1996,109:542-546.
67 朱雷明，蔡端，张延龄．模拟胆汁中游离胆红素的热力学研究．外科理论与实践1999，4：13-17．
68 Nakai K, Tazuma S, Ochi H, et al. Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influence of bilirubin on bile lithogenicity. Biochim Biophys Acta 2001, 1534:78-84.
69 Smith B, La Mont Yr. Identification of gallstone mucin-bilirubin complex in human cholesterol gallstone matrix. J Clin Invest 1985,76: 439-445.
70 Binette JP, Binette MB. The proteins and the formation of gallstones. Clin Chim Acta 2000,296: 59-69.
71 JaeWoon C, Klinkspoor JH, Tadashi Y, et al. Lipopolysaeeharide from Esehrichia coli stimulates mucin secretion by cultured dog gallbladder epithelial cells.Hepatology 1999, 29:1352-1357.
72 Goscinski G, Lundholm M, Odenholt I, et al. Variation in the propensity to release endotoxin after eefuroxime exposure in different gram-negative bacteria:uniform and dose dependent reduction by addition oftobramycin. Scan J Infect Dis 2003,35:40-46.
73 邓学稼，蔡端，张延龄，等．利胆冲剂防治胆石症的临床和实验研究．中华消化杂志1982，2：10-13．
2． Maki T. Pathogenesis of calcium bilirubinate gallstone: role of E. coli, beta-glucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation. Ann Surg 1966,164: 90-100．
3． Strasberg SM,Clavien PA, Harvey PR. Pathogenesis of cholesterol gallstones. HPB Surg 1991,3:79-102
4． Hatsushika S, Tazuma S, Kajiyama G. Nucleation time and fatty acid composition of lecithin in human gallbladder bile. Scand J Gastroenterol 1993,28:131-136．
5． Moser AJ, Abedin MZ, Roslyn JJ. Increased biliary protein precedes gallstone formation. Dig Dis Sci 1994,39:1313-1320．
6． Luk As, Kaler EW,Lee SP. Protein lipid interaction in bile: effects of biliary proteins on the stability of cholesterol-lecithin vesicles. Biochim Biophys Acta 1998,1390: 282-292．
7． Mansour A, Dawoud I, Gad-El-Hak N. The potential site of disordered gallbladder contractility during the early stage of cholesterol gallstone formation. Hepatogastroenterology 1998,45:1404-1409．
8． Truedson H, Elmros T, Holm S. The incidence of bacteria in gallbladder bile at acute and elective cholecystectomy. Acta Chir Scand 1983,149:307-313．
9． Moore EW. Biliary calcium and gallstone. Hepatology 1990,12:206s-214s.
10 Kaufman HS, Magnuson TH, Pitt HA, et al. The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown pigment gallstones. Hepatology 1994,19: 1124-1132．
11 Okido M, Shimizu S, Ostrow JD, et al. Isolation of a calcium-regulatory protein from black pigment gallstones: similarity with a protein from cholesterol gallstones. Hepatology 1992,15: 1079-1085．
12 Swidsinsky A, Ludwig W, Pahlig H, et al. Molecular genetic evidence of bacterial colonization of cholesterol gallstones. Gastroenterology 1995,108: 860-864．
13 Weisberg WG, Barns SM, Pelletier DA, et al. 16S ribosomal DNA amplification for phylogenetic study. 1991,173: 697-703．
14 Greisen K, Loeffelholz M, Purohit A, et al. PCR primes and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clinic Microbiology 1994,32: 335-351．
15 Lee DK, Tarr P, Haigh WG, et al. Bacterial DNA in mixed cholesterol gallstones. Am J Gastroenterol 1999,94:3502-3506．
16伍晓汀，肖路加，黎介寿，等．由胆固醇结石提取DNA进行套式聚合酶链反应的研究．中华外科杂志1997，35：663-666．
17 Soloway RD, Crowther RS. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis. Gastroenterology. 1995,108: 934-936．
18 Swidsinski A, Lee SP. The role of bacteria in gallstone pathogenesis. Front Biosci 2001,6:E93-E103．
19 Cariati A, Cetta F. Re: Kawai et al.-Bacteria are not important in the formation of pure cholesterol stones. Am J Gastroenterol 2002,97:2921-2923．
20 Swidsinski A, Khilkin M, Pahlig H, et al. Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstone. Hepatology 1998,27:662-665．
21 Fromm H, Amin P, Klein H, et al. Use of a simple enzymatic assay for cholesterol analysis in human bile. J Lipid Res 1980,21:259-261．
22 Gurantz D, Laker MF, Hoffmann AF. Enzymatic measurement of choline-containing phospholipids in bile. J Lipid Res 1981,22:373-376．
23 Turley SD, Dietschy JM. Re-evaluation of the 3 a-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978; 19:924-928．
24 Carey MC. Critical tables and cholesterol saturation of bile. J Lipid Res 1978,19:945-955．
25 Ohdan H, Oshiro H, Yamamoto Y, et al. Bacteriological investigation of bile in patients with cholelithiasis. Surgery Today 1993,23:390-395．
26 Fox JG, Dewhirst FE, Shen Z, et al. Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology, 1998,114:755-763．
27 Figura N, Cetta F, Angelico M, et al. Most Helicobacter pylori-infected patients have specific antibodies, and some also have H.pylori antigens and genomic material in
bile. Is it a risk factor forgallstone formation? Dig Dis Sci, 1998, 43: 854-862.
28 Kawai M, Iwahashi M, Uchiyama K, et al. Gram-positive cocci are associated with the formation of completely pure cholesterol stones. Am J Gastroenterol 2002, 97:83-88.
29 Prouty AM, Schwesinger WH, Gunn JS. Biofilm formation and interaction with the surfaces of gallstones by Salmonella spp. Infect Immun, 2002, 70: 2640-2649.
30 韩文胜，石景森，卓健生，等．检测胆囊结石胆汁中L型细菌的研究及其意义．中华实验外科杂志2002，18：207-208．
31 吕厚东，曹卉，山长武，等．人及多种动物胆汁诱导出细菌L型的实验研究．济宁医学院学报1995，18：22-23．
32 Goodhart GL, Levison ME, Trotman BW, et al. Pigment vs cholesterol cholelithiasis.Bacteriology of gallbladder stones, bile, and tissue correlated with biliary lipid analysis. Am J Dig Dis 1978; 23:877-882.
33 黄美雄，朱明炜，宋京海，等．胆道感染细菌的变迁及细菌对抗生素敏感性分析．中华肝胆外科杂志2001，7：272-274．
34 朱雷明，陈为欢，蔡端，等．T管胆汁细菌培养及对抗生素敏感性分析．外科理论与实践经验2003，8：124-126．
35 Wetter LA, Hamadeh RM, Griffiss JMCL, et al. Difference in outer membrane characteristics between gallstone associated bacteria and normal bacteria flora. Lancet 1994,343:444-447.
36 Monstein HJ, Jonsson Y, Zdolsek J, et al. Identification of Helicobacter pylori DNA in human cholesterol gallstone. Scand J Gastroenterol 2002,37:112-119.
37 Myung SJ, Kim MH, Shim KN, et al. Detection of Helicobacter pylori DNA in human biliary tree and its association with hepatolithiasis. Dig Dis Sci 2000,45:1405-1412.
38 La Scola B, Barrassi L, Raoult D. Isolation of new fastidious alpha proteohacteria and Afipia felis from hospital water supphes by direct plating and amoebal co-culture procedures. FEMS Microbiol Ecol 2000.34:129-137.
39 Harvey PR, Upadhya GA, Strasberg SM,. Immunoglobulins as nucleation proteins in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 1991,266:13996-14003．
40 Abei M, Schwarzendrube J, Nuutinnen H, et al. Cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, a 1-acid glycoprotein, phospholipase C,and aminopeptidase N. J Lipid Res 1993,34:1141-1148．
41 Miquel JF, Nunez L, Rigotti A, et al. Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles. FEBS Lett 1993,318:45-49．
42 de Brujin MAC, Mok KS, Out T, et al. Immunoglobulins and a 1-acid glycoprotein do not contribute to the cholesterol crystallization-promoting effect of Concanavalin A-binding biliary protein. Hepatology 1994,20: 626-632．
43 Alissa K, Saunier P, Russo M, et al. Neonatal cholestatic lithiasis associated with E. coli infection. Arch pediatr 1996,3: 144-146．
44 Kibe A, Dudley MA, Halpem DZ, et al. Factors influencing cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile. J Lipid Res 1985, 26:1102-1111．
45 Holan KR, Holzbach RT, Hermann RE, et al. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979, 77:611-617．
46 de 1a porte PL, Lafont H, Domingo N, et al. Composition and immunofluorescence studies of biliary "sludge" in patients with cholesterol or mixed gallstones. J Hepatol 2000,33:352-360．
47王巍，韩天权，张圣道．胆固醇结石病胆汁游离脂肪酸的水平及其临床意义．中华外科杂志1995，33：659-661．
48 Halpem Z, Goldman G, Peled Y, et al. Free fatty acids have nucleating effects in model biles. Liver 1992,12:107-111．
49 Kabongo Muamba ML. Exoenzymes of Propionibacterium acnes. Can J Microbio 1982,28:758-761．
50 Nakamara T, Taniguchi H, Takeuchi K, et al. Phospholipase C of an oral strain of Propionibacterium. Purification and partial characterization. Zbl Bakt Hyg A 1984,257:20-26．
51 Pattinson NR, Willis KE. Effect of phospholipase C on cholesterol solubilization in model bile. Gastroenterology 1991, 101: 1339-1344.
52 Lichtenberg D, Ragimova S, Peled Y, et al. Phospholipid peroxidation as a factor in gallstone pathogenesis. FEBS Lett 1988,228:179-181.
53 马保金，蔡端，张延龄，等．胆汁泡蛋白成核活性及其氨基酸序列分析的研究．外科理论与实践1999，4：22-23．
54 项建斌，蔡端，张延龄，等．33.5kDa胆汁泡蛋白促成核活性及其对泡形态学影响的研究．肝胆胰外科杂志1999，1：8-10．
55 Miguel JF, von Ritter C, Del Pozo R, et al. Fibronectin in human gallbladder:cholesterol pronucleating and/or "link" protein? Am J Physiol 1994, 267: G393-400.
56 焦宛，张延龄，蔡端，等．胆汁促成核糖蛋白的分离提纯及理化性质研究．中华外科杂志1994，32：271-272．
57 励峰，蔡端，莫汉庆，等．ELISA法检测200kDa糖蛋白．中华消化杂志1997，17：333-335．
58 Miquel JF, Rigotti A, Rojas E, et al. Isolation and purifiaction of human biliary vesicles with potent cholesterol-nucleation-promoting activity. Clin Sci 1992, 82:175-180.
59 Miquel JF, Nunez L, Amigol L, et al. Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile.Gastroenterology 1998,114: 1016-1023.
60 项建斌，蔡端，张延龄，等．胆汁泡蛋白促成核活性以及在不同人群含量的分布．中华实验外科杂志2001，1：83．
61 Ostrow JD, Celic L, Mukerjee P, et al. Molecular and micellar associations in the pH-dependent stable and metastable dissolution ofunconjugated bilirubin by bile salts. J Lipid Res 1988,29: 335-348.
62 Fu XP, Zhou XS, Zhang WH, et al. "Bilirubin-calcium compound" precipitation and the effect ofbile salts on it;the pathogenesis of pigment gallstone. Chinese Med J 1998,728-738.
63 Leung JW, Liu YL, Leung PS, et al. Expression of bacterial beta-glucuronidase in human bile: an in vitro study. Gastrointest Endose 2001,54:346-350.
64 蔡端，张延龄，邬建国，等．胆汁中胆固醇和胆红素含量的相关性研究．上海医学1989，12：709-711．
65 Kaufman HS, Magnuson TH, Webb TH, et al. Bilirubin monoglucuromde promotes cholesterol gallstone formation. J Surg Res 1991,50:504-509.
66 Zhu XG, Tu XQ, Wang RH, et al. Observation on solubility and pro-nucleative activity of monoconjugated bilirubin. Chinese Med J 1996,109:542-546.
67 朱雷明，蔡端，张延龄．模拟胆汁中游离胆红素的热力学研究．外科理论与实践1999，4：13-17．
68 Nakai K, Tazuma S, Ochi H, et al. Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influence of bilirubin on bile lithogenicity. Biochim Biophys Acta 2001, 1534:78-84.
69 Smith B, La Mont Yr. Identification of gallstone mucin-bilirubin complex in human cholesterol gallstone matrix. J Clin Invest 1985,76: 439-445.
70 Binette JP, Binette MB. The proteins and the formation of gallstones. Clin Chim Acta 2000,296: 59-69.
71 JaeWoon C, Klinkspoor JH, Tadashi Y, et al. Lipopolysaeeharide from Esehrichia coli stimulates mucin secretion by cultured dog gallbladder epithelial cells.Hepatology 1999, 29:1352-1357.
72 Goscinski G, Lundholm M, Odenholt I, et al. Variation in the propensity to release endotoxin after eefuroxime exposure in different gram-negative bacteria:uniform and dose dependent reduction by addition oftobramycin. Scan J Infect Dis 2003,35:40-46.
73 邓学稼，蔡端，张延龄，等．利胆冲剂防治胆石症的临床和实验研究．中华消化杂志1982，2：10-13．