模拟胆汁体系中胆固醇结石内细菌对胆固醇晶体形成的作用

目的 探讨胆固醇结石中的细菌与结石形成的关系。方法 观察胆固醇结石中的细菌在模拟胆汁中的生长活性及其对胆固醇晶体成核时间(NT)的影响。结果 (1)模拟胆汁中大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、粪肠球菌、产孢梭菌和艰难梭菌生长旺盛;痤疮丙酸杆菌长势偏弱;脆弱类杆菌生长受抑制。(2)仅铜绿假单胞菌和粪肠球菌能缩短胆固醇晶体成核时间。(3)加入铜绿假单胞菌或粪肠球菌的胆汁,晶体形成呈现缓慢的阶段性演变过程。结论 铜绿假单胞菌和粪肠球菌在模拟胆汁中具有促成核活性,而痤疮丙酸杆菌无促成核作用。     

细菌促胆固醇晶体成核的机制研究

目的 定位铜绿假单胞茼、粪肠球菌促胆固醇晶体成棱活性来源,并探讨其作用途径.方法 分离细菌的外分泌物、细胞破坏后的上清和沉淀裂解组分.观察其在模拟胆汁体系中对胆固醇晶体形成的时间的影响,并对活性组分进行蛋白质提纯及促成核活性现察.结果 ①粪肠球菌和铜绿假单胞菌的外分泌组分和破壁上清组分能缩短胆固醇晶体成核时间(nucleation time,NT),破壁后沉淀物的裂解成分未见此活性.②大肠杆菌的外分泌组分不能缩短胆固醇晶体成核时间,但是其破壁上清组分能缩短胆固醇晶体成核时间.③从外分泌组分和破壁上清组分中提纯的蛋白质仍具有缩短胆固醇晶体成核时间的能力.结论 粪肠球菌和铜绿假单胞菌的外分泌组分和破壁上清组分,大肠杆菌的破壁上清组分具有促进胆固醇晶体形成的能力.蛋白质是重要的活性成分.     

细菌对模拟胆汁热力学平衡的影响

目的:探讨细菌对模拟胆汁物理鄄化学平衡的影响。方法:观察加入大肠杆菌、粪肠球菌、铜绿假单胞菌和生理盐水的模拟胆汁中胆固醇晶体形成的时间和胆汁成分析出黏附的时间,测量胆汁黏度值和胆汁类脂成分溶解度的变化。结果:加入粪链球菌或铜绿假单胞菌的模拟胆汁,黏度值增加,试管壁上有黏附物质析出。加入大肠杆菌或生理盐水的模拟胆汁中,无类似变化。在不同的胆固醇饱和条件下,黏附物质析出的时间早于或等于胆固醇晶体形成的时间。胆固醇溶解度在加入粪链球菌或铜绿假单胞菌的模拟胆汁中,低于在加入大肠杆菌或生理盐水的模拟胆汁;胆汁酸盐或磷脂的溶解度无显著差异。胆汁析出的黏附物质中胆固醇是主要成分之一。结论:粪链球菌或铜绿假单胞菌增加模拟胆汁的黏度并有黏附物质形成,而降低胆固醇的溶解度。     

胆固醇结石中细菌对人胆汁胆固醇晶体形成的作用

目的探讨胆固醇结石中的细菌与结石形成的关系。方法观察胆固醇结石中的细菌在人胆汁中的生长情况及其对胆固醇晶体形成的影响。结果(1)人胆汁中大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、无乳链球菌和脆弱类杆菌生长旺盛；痤疮丙酸杆菌、产孢梭菌和艰难梭菌生长缓慢；粪肠球菌生长受抑制。(2)在未离心的人胆汁中，大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、无乳链球菌、粪肠球菌和脆弱类杆菌明显缩短胆固醇晶体成核时间(NT)，产生的最大晶体量高于无菌的对照胆汁，在离心后的胆汁中该作用消失；痤疮丙酸杆菌、产孢梭菌和艰难梭菌在离心或未离心的胆汁中均不能显著降低NT，但是后两者在未离心的人胆汁中产生的最大晶体量高于对照胆汁。(3)人胆汁离心后的沉淀中含有蛋白质。结论(1)不同细菌在人胆汁中的生存能力有差异。(2)大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、无乳链球菌、粪肠球菌和脆弱类杆菌在未离心的人胆汁中有促成核作用。(3)离心后人胆汁中某些蛋白质的丢失可能与细菌促成核能力的丧失有关。(4)痤疮丙酸杆菌无促成核作用。     

综合模拟胆汁体系中未结合胆红素与胆固醇的溶解方式

目的:在含有未结合胆红素(UCB)、胆固醇、磷脂和胆盐的综合模拟胆汁体系中,探讨UCB与胆固醇的溶解方式及相互关系.方法:检测UCB在Small模拟胆汁、胆汁泡(胆固醇-磷脂泡)溶液、泡对照组和本底对照组中的饱和溶解度,并分析比较Small模拟胆汁在加入过量UCB保存、过滤前后胆固醇含量的变化;同时观察UCB对胆固醇过饱和胆汁成核时间(NT)的影响.结果:微胶粒、泡能结合UCB而使其溶解,UCB能促进胆固醇结晶形成、NT缩短,且其差异显著.结论:UCB溶解依赖胆固醇的溶解方式,同时又可能作为促成核因子参与胆固醇结石形成.     

T管胆汁细菌培养及对抗生素敏感性分析

目的：了解临床已无感染症状病人的T管引流胆汁的细菌感染情况及对抗生素的敏感性。方法：对2001年12月至2002年12月116例术后已无感染症状病人的T管引流胆汁的细菌培养和抗生素敏感性试验结果作统计分析。结果：T管胆汁细菌培养的阳性率为59．91％。158株微生物中包括23种细菌和3种真菌。肺炎克雷伯菌、铜绿假单胞菌、大肠埃希菌、表皮葡萄球菌、金黄色葡萄球菌、粪肠球菌和尿肠球菌占胆道细菌的72．75％，24例混合感染中13例感染铜绿假单胞菌，同时发现嗜麦芽窄食单胞菌和鲍曼不动杆菌等条件致病菌感染。细菌对头孢类抗生素具有较高的耐药率，对亚胺培南、万古霉素、阿米卡星有较高的敏感性。在不同病人相同菌种的药敏试验结果有较大差异，提示菌株不同。结论：临床无感染症状病人的T管引流胆汁具有较高的细菌感染率，二重感染和耐药菌株的增加是一旦发生胆汁性腹膜炎时严重感染的基础。     

模拟胆汁体系中游离胆红素的热力学研究

在Small的胆汁热力学体系基础上建立一个包含游离胆红素(UCB)在内的更完善的热力学体系。方法:研究胆汁中胆固醇、磷脂、胆汁酸盐、pH和总脂浓度等因素对UCB溶解平衡的影响,制作UCB在胆固醇饱和指数(CSI)1.0的模拟胆汁中的饱和溶解度表,并建立描述UCB饱和程度的指标,评估其临床意义。结果:磷脂能抑制UCB溶解;胆固醇则增溶UCB,但随着胆固醇饱和指数的增高,UCB溶解度又呈反比例下降;总脂浓度(TL)越高,UCB的溶解度也越高;在Carey表格的基础上,任何一份饱和模拟胆汁中(pH 7.0,TL<10g/dl),均可找到一个确定的UCB饱和溶解值。临床上胆石症患者与非胆石症患者的游离胆红素饱和指数(BSI)有显著差异(P<0.05)。结论:(1)UCB和胆固醇有共同沉淀的热力学基础。(2)建立一个UCB饱和溶解度表(pH 7.0,CSI 1.0),BSI在致石胆汁和非致石胆汁之间有显著差异。     

大血藤、牡丹皮、赤芍有效组分间对肠道致病菌体外抑菌的协同作用

目的:观察大血藤、牡丹皮及赤芍有效组分合用对四种腹腔感染细菌的协同抑制作用.方法:提取大血藤中绿原酸,牡丹皮中丹皮总苷和赤芍中的赤芍总苷三种抑菌组分,定量后以大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、粪肠球菌为实验菌株,琼脂二倍稀释法和棋盘试验法评价三种抑菌组分单用与联用的体外抑菌效果.结果:三种组分单用均对四种细菌的标准株及临床株有抑菌效果,其中绿原酸对大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌和粪肠球菌的MIC分别为8.5μg/mL、34 μg/mL、8.5 μg/mL、2.125 μg/mL,丹皮总苷对四菌株的MIC分别为86 μg/mL、344μg/mL、21.5 μg/mL、86 μg/mL,赤芍总苷对四菌株的MIC分别为117.5 μg/mL、470 μg/mL、117.5 μg/mL、117.5 μg/mL;绿原酸与丹皮总苷合用对铜绿假单胞菌和粪肠球菌有部分协同作用,丹皮总苷和赤芍总苷合用对粪肠球菌有部分协同,绿原酸与赤芍总苷合用对四种细菌均为无关作用.结论:所提取的三种组分均具有抑菌活性,合用后抑菌活性增强.     

胆道疾病患者胆汁病原菌谱与耐药监测

目的 了解胆道疾病患者胆汁中病原菌的分布和耐药情况,为临床提供病原学诊断和合理使用 抗菌药物的依据。方法 回顾性分析武汉大学人民医院2016年1月至2018年12月临床胆汁标本中分离的 病原菌,通过Phoenix-100全自动微生物鉴定药敏系统进行鉴定和药敏分析。结果 共分离出1 251株病原 菌,其中革兰氏阳性菌504株（40.29%）、革兰氏阴性菌685株（54.75%）、真菌 62株（4.96%）。前 6位病原菌 依次为大肠埃希菌、屎肠球菌、粪肠球菌、肺炎克雷伯菌、铜绿假单胞菌、阴沟肠杆菌,分别占19.90%、 16.55%、8.79%、8.31%、3.84%、3.52%。大肠埃希菌和肺炎克雷伯菌的超广谱β-内酰胺酶（ESBLs）检出 率分别为44.98%和18.27%。碳青霉烯类耐药的大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌的检出率为1.20%、 3.85%和6.82%。碳青霉烯类耐药的鲍曼不动杆菌和铜绿假单胞菌检出率分别为63.64%和45.83%。结论 胆道感染以肠杆菌科细菌和肠球菌属细菌为主要病原菌。碳青霉烯类耐药的细菌应引起临床高度重视,根据药敏结果合理选用抗生素。     

外科感染患者细菌分布及耐药性的单中心研究

目的 分析外科感染患者细菌分布及其对常用抗菌药物的耐药性,为外科感染的规范化治疗提供依据.方法 回顾性调查分析2008年1月至201 1年12月外科感染患者送检标本的细菌鉴定及药物敏感性检测结果.结果 3257份临床标本共分离菌株3829株,革兰阴性杆菌占62.4％(以大肠埃希菌、铜绿假单胞菌和肺炎克雷伯菌为主);革兰阳性球菌占37.6％(以肠球菌、金黄色葡萄球菌及凝固酶阴性葡萄球菌为主),其中金黄色葡萄球菌、粪肠球菌检出率呈升高趋势.大肠埃希菌及肺炎克雷伯菌对亚胺培南、阿米卡星、哌拉西林/他唑巴坦等抗菌药物耐药率较低;铜绿假单胞菌和鲍曼不动杆菌对头孢类、碳青霉烯类及喹诺酮类抗菌药物耐药率较高,呈多药耐药性;所有葡萄球菌、粪肠球菌对万古霉素和替考拉宁敏感(100％),但耐万古霉素屎肠球菌检出率呈上升趋势(1.9％～7.5％).产超广谱β-内酰胺酶(ESBL)大肠埃希菌检出率为45.6％ ～61.5％;产ESBL肺炎克雷伯菌检出率呈波动表现;耐甲氧西林金黄色葡萄球菌检出率较高(21.1％ ～55.8％),耐甲氧西林表皮葡萄球菌检出率明显高于其他阳性球菌.结论 我院外科临床感染病原菌以革兰阴性杆菌为主,临床分离细菌耐药现象较为普遍,铜绿假单胞菌和鲍曼不动杆菌药物耐药率较高.     

Supersaturation of canine gallbladder bile with calcium bilirubinate during formation of pigment gallstones

Analogous to cholesterol gallstones forming in bile supersaturated with cholesterol, pigment gallstones may form in bile supersaturated with calcium bilirubinate. We tested this hypothesis in a dietary model of pigment gallstones. The concentration of ionized calcium (Ca++) and unconjugated bilirubin (UCB) was measured in 15 normal dogs and in 15 dogs with pigment gallstones induced by 6 weeks of a methionine-deficient diet. Although there was minimal change in the gallbladder's ability to acidify or concentrate bile, both [Ca++] and [UCB] markedly increased. These values were compared with equilibrium concentrations in model bile solutions. In all normal bile, the [UCB] was equal to or lower than the mean [UCB] concentration of model bile solutions with comparable [Ca++]. However, in all but one bile sample from dogs with pigment gallstones, the [UCB] exceeded this concentration and was therefore supersaturated with calcium bilirubinate. This supports the hypothesis that calcium bilirubinate precipitation is important in the formation and growth of pigment gallstones.     

Altered bile composition during cholesterol gallstone formation: cause or effect?

Gallbladder stasis, increased gallbladder absorption, and elevated biliary levels of calcium, hydrogen ion, and bilirubin have been implicated as factors potentially critical to cholesterol crystal precipitation. Previous studies, however, have analyzed bile only when crystals or gallstones have already formed. Therefore, we tested the hypothesis that changes in bile composition are a late effect, occurring only after crystal formation. Adult male prairie dogs were fed a standard nonlithogenic control diet (n = 7) or a lithogenic 1.2% cholesterol diet for 5, 9, or 14 days to cause cholesterol saturation (n = 7), cholesterol monohydrate crystals (n = 7), or gallstones (n = 7). Gallbladder bile was examined microscopically for crystals, and analyzed for ionized calcium, bilirubin, pH, total protein, and biliary lipids. The ratio of gallbladder to hepatic bile radiolabeled cholic acid specific activity (Rsa) was calculated as an index of gallbladder stasis. Cholesterol saturation index was calculated. The results demonstrate that increased gallbladder bile cholesterol saturation and total protein concentration precede cholesterol monohydrate crystal precipitation. However, changes in gallbladder ionized calcium, unconjugated bilirubin, pH, stasis, and absorption were noted only after crystals and gallstones had already formed. These data indicate that alterations in gallbladder bile calcium, bilirubin, pH, stasis, and absorption are not early changes, but occur simultaneously with or after crystal formation. Increased biliary protein, however, which was elevated prior to nucleation, may be an important mediator of cholesterol precipitation in cholesterol-saturated bile.     

Clinical and experimental studies on the formation of calcium bilirubinate gallstones

We identified the bacteria in the bile and measured the activity of bacterial beta-glucuronidase and analyzed the percentage of bilirubin glucuronide and unconjugated bilirubin by high performance liquid chromatography in the bile of human biliary stone disease. The percentage of positive bacterial infection in the bile are 72.0% with calcium bilirubinate gallstone, and 42.3% with cholesterol gallstone. The activity of beta-Glucuronidase (U/dl.hr.) was significantly higher in the bile of calcium bilirubinate gallstone than that of cholesterol gallstone (7013 +/- 5113 vs 3338 +/- 2615, mean +/- S.D.). Also, the percentage of unconjugated bilirubin (IX alpha) of the bile was significantly higher in calcium bilirubinate gallstone than in cholesterol gallstone (5.7 +/- 4.7% vs 2.6 +/- 2.0%, mean +/- S.D.). The beta-Glucuronidase activity of bacteria was as follows; E. coli 18752, K. pneumoniae 333, E.cloacae 124, S.faecalis 324, and B.fragilis 983. After 60 minutes' incubation at 37 degrees C of normal bile with E.coli, the percentage of unconjugated bilirubin (IX alpha) increased from 1.1% to 9.1%. Whereas, the other four bacteria did not increase the unconjugated bilirubin at all. We confirmed that the increase of unconjugated bilirubin caused by the high bacterial beta-Glucuronidase activity was the most important factor in the formation of calcium bilirubinate gallstone.     

急性胆道感染大鼠肝细胞原胆汁分泌的改变

目的:观察急性胆道感染大鼠肝细胞原胆汁分泌的变化.方法:制作急性胆道感染大鼠动物模型,取其肝细胞进行原代培养,利用反向高效液相色谱(RP-HPLC)技术,检测肝细胞培养上清液中胆汁酸和UCB含量.结果:急性胆道感染大鼠肝细胞培养上清液中均可检出一定量的非结合胆红素(UCB),同时三种主要结合胆汁酸(GCA,GCDCA,TCA)分泌减少,与对照组比较,有显著性差异(P＜0.01).结论:急性胆道感染大鼠肝细胞分泌会发生明显改变,原胆汁分泌胆汁酸减少,胆色素代谢发生改变,对胆色素结石的形成具有一定的影响.     

肝硬化胆囊结石患者的胆囊胆汁和结石成分分析

目的: 通过测定肝硬化患者胆囊胆汁和结石成分,分析胆囊结石的类型及其与胆汁成分改变的关系. 方法: 将研究对象分四组,即非胆囊结石对照组(A组,13例)、单纯胆囊结石组(B组,16例)、肝硬化组(C组,5例)和肝硬化胆囊结石组(D组,7例).测定A组13例、B组15例、C组5例和D组7例的胆囊胆汁成分.对B组16例、D组6例行结石成分分析,用肉眼和实体显微镜观察结石的表面及剖面,并按1984年傅培彬等人提出的结石分类法分类,结石成分用化学方法分析. 结果: ①两组肝硬化的胆汁中未结合胆红素(UCB)含量明显升高,胆汁酸(TBA)含量明显降低,与对照组比较差异有显著性意义(P<0.05).结石中的66.7%为黑结石.单纯胆囊结石组胆汁中胆固醇(TC)含量明显升高,胆汁酸含量明显降低,与对照组比较差异有显著性意义(P<0.05).结石中的87.5%为胆固醇型结石.②两组肝硬化(C和D组)的血清TB和UCB均显著高于A和B两组(P<0.05).而血清TBA在B、C和D组中却明显低于A组(P<0.05).血TC在C和D两组低于A和B两组(P<0.05). 结论: 肝硬化患者好发胆囊胆色素结石,且以黑色结石多见,这与肝硬化时肝脏代谢胆红素和胆固醇障碍胆汁中UCB升高、TBA降低有关.     

Changes in bile components during the formation and prevention of black pigment gallstone in a guinea pig model

Black pigment gallstones were found in 22 out of 23 guinea pigs one week after the common bile ducts were partially ligated (S group). The incidence decreased to 11/20 (P less than 0.01) if the animals were fed with a chow containing bile salt mixture, glucurolactone, ane aspirin (S+M group). Three weeks after the ligation the incidence of gallstone in S group and S+M group was 17/18, and 9/10, respectively (P greater than 0.05). Glucuronidase (beta-G) activity in the bile of S group was not higher than that of control group (C group, P greater than 0.05), and all the bile samples were sterile. The biliary concentrations of ionized calcium (ICa), unconjugated bilirubin (UCB), total calcium (TCa), total bilirubin (TBr), glycoprotein (GIy. P), and PH significantly fluctuated among guinea pig groups and were parallel to gallstone incidence. The results of this study could hardly be explained by Maki beta-G theory, but were consistent with the assumption that the precipitation-dissolution equilibrium of calcium bilirubinate is the key of pigment gallstone formation, thus the increases of the above mentioned bile components, including beta-G activity, would bias this equilibrium towards calcium bilirubinate precipitation and therefore promote gallstone formation. On the contrary, stone formation would be prevented.