胆道癌、胆石症患者胆汁标本菌群分析

目的 探讨胆石症发病与细菌感染的关系.方法采用需氧、专性厌氧和微需氧方法 对择期手术治疗的胆道癌、胆石症患者的胆汁标本进行细菌分离培养、API鉴定和菌群分析.结果 胆石症患者细菌阳性分离率为36%（34/95）,胆石症合并胆道癌患者为43%（6/14）,胆道癌患者为57%（8/14）;色素型胆石症患者胆汁细菌阳性分离率为77%（10/13）,混合型胆石症患者为29%（16/56）,胆固醇型胆石症患者为23%（3/13）,不明胆石类型胆石症患者为45%（10/22）.共分离出69株菌株,其中肠杆菌科细菌30株,肠球菌属细菌19株,其余为葡萄球菌属细菌、链球菌属细菌等.结论 胆石症患者胆汁中分离出细菌,且以色素型胆石症患者胆汁标本细菌阳性分离率最高,胆汁细菌分布又以肠杆菌科细菌和肠球菌属为主,提示色素型胆石症形成与细菌有一定关系.     

细菌rDNA分类鉴定的方法学研究

目的 研究细菌rDNA快速鉴定方法.方法 以细菌16S rDNA、16S-23S rDNA基因间隔区（ISR）和常见细菌耐药基因为对象,通过改进标本中细菌DNA提取方法,指纹分析、直接测序与多重聚合酶链反应（PCR）技术,建立快速细菌分子鉴定方法.结果 细菌属种不同表现出独特的16S-23S rDNA ISR指纹,在分析软件指导下进行初步细菌鉴定和亲缘关系研究;16S rDNA和16S-23SrDNA ISR序列可确定细菌种与型;序列分析与生化鉴定一致.多重PCR可解决葡萄球菌16S rDNA与mecA基因、产超广谱β内酰胺酶菌16S rDNA与TEM和SHV基因同步检测.结论 细菌rDNA分类方法,提高了非培养细菌的鉴定能力.     

胆宁片对胆汁33.5kDa泡蛋白含量和结构的影响

目的：探讨胆宁片对胆石症患者胆汁中33.5kDa泡蛋白含量及其结构的影响。方法：2008年1月—2010年3月对60例胆囊切除病例和40例胆总管探查病例进行前瞻性临床研究,其中60例胆囊切除病例随机分为3组：胆固醇结石胆宁片组（n=20）、胆固醇结石对照组（n=20）、胆色素性结石组（n=20）,另外设立正常对照组（n=20）。通过酶联免疫吸附法检测每份胆汁中33.5kDa泡蛋白含量,比较各组之间的差异。40例胆总管探查病例分为研究组和对照组（n=20）,通过蔓陀螺凝集素探针（DSA）介导的蛋白印迹法检测胆汁中33.5kDa泡蛋白糖链条带DSA结合率,比较2组糖链结构的变化。结果：胆固醇结石组胆汁中33.5kDa泡蛋白含量显著高于胆色素结石组和正常对照组,胆固醇结石胆宁片治疗组胆汁33.5kDa泡蛋白含量较对照组显著降低（P〈0.01）。胆固醇结石治疗组泡蛋白糖链条带DSA结合率较对照组显著下降（P〈0.05）。结论：胆宁片能降低胆固醇结石患者胆汁中33.5kDa泡蛋白含量,并通过改变糖链结构而使其促成核活性发生变化。     

胆囊胆固醇结石患者肝脏LRH－1及调控基因蛋白表达差异

目的研究LRH－1及其调控的靶基因ABCG5／8、CETP、SRBI和CYP7A1蛋白表达变化与胆囊胆固醇结石形成的关系。方法收集胆囊胆固醇结石患者20例,无结石（肝良性肿瘤,肝脏外伤,胆囊非胆固醇息肉）对照15例,分别测定血清血脂全套,胆汁成分分析,结石成分分析,免疫组化法研究胆石患者肝脏LRH一1及其调控的靶基因：SRBI、ABCG5／8和CYP7A1的蛋白表达。结果胆囊胆固醇结石患者胆汁胆固醇饱和指数高于对照组（P〈0．01）。免疫组化分析结果提示结石组肝脏核受体LRH－1蛋白表达明显高于对照组（P〈0．05）,结石组肝脏SRBI、ABCG5／8和CYP7A1蛋白表达也明显高于对照组C〈0．05）。结论核受体LRH-1及其调控的靶基因SRBI和ABCG5／8可能与胆囊胆固醇结石形成相关。     

不同分期矽肺患者气道细菌群落微生态研究

目的 探讨不同分期矽肺患者气道细菌微生物构成的差异。方法 采用立意抽样方法，选择29例职业性矽肺病患者，13例健康受试者为研究对象，其中矽肺壹期患者6例（矽肺壹期组），矽肺贰期患者15例（矽肺贰期组），矽肺叁期患者8例（矽肺叁期组）。采集42例受试者痰液样本，采用16S r DNA测序技术检测痰液样品中细菌微生物，分析4组受试者细菌微生物的相对丰度差异，对健康受试者与矽肺患者组间存在差异的细菌微生物进行物种多样性指数（alpha多样性指数、beta多样性指数）差异分析；绘制受试者工作特征曲线（ROC）评估细菌微生物作为生物标记物的可行性。结果 健康受试者与矽肺患者痰液样品中奈瑟菌属（Neisseria）、莫拉氏菌属（Moraxella）、葡萄球菌属（Staphylococcus）、棒状杆菌属（Corynebacterium）、狡诈球菌属（Dolosigranulum）、密螺旋体菌属（Treponema）相对丰度分别比较，差异均有统计学意义（P <0.05）。LEfSe分析结果显示，矽肺壹期组增加的物种包括Neisseria、奈瑟氏菌科（Neisseriaceae）、假单胞菌目（P...     

胆道癌、胆石症患者胆汁标本菌群分析

目的探讨胆石症发病与细菌感染的关系。方法采用需氧、专性厌氧和微需氧方法,对择期手术治疗的胆道癌、胆石症患者的胆汁标本进行细菌分离培养、API鉴定和菌群分析。结果胆石症患者细菌阳性分离率为36%(34/95),胆石症合并胆道癌患者为43%(6/14),胆道癌患者为57%(8/14);色素型胆石症患者胆汁细菌阳性分离率为77%(10/13),混合型胆石症患者为29%(16/56),胆固醇型胆石症患者为23%(3/13),不明胆石类型胆石症患者为45%(10/22)。共分离出69株菌株,其中肠杆菌科细菌30株,肠球菌属细菌19株,其余为葡萄球菌属细菌、链球菌属细菌等。结论胆石症患者胆汁中分离出细菌,且以色素型胆石症患者胆汁标本细菌阳性分离率最高,胆汁细菌分布又以肠杆菌科细菌和肠球菌属为主,提示色素型胆石症形成与细菌有一定关系。     

16S rDNA基因芯片检测临床常见感染性细菌

目的 提高细菌和临床微生物检测的速度和准确性，建立含有20种细菌探针在内的感染性细菌检测用基因芯片模型。方法 使用16S rDNA克隆探针和合成的寡核苷酸探针两种，利用点样仪制成基因芯片。细菌DNA经过16S rDNA通用引物扩增后与芯片上的探针杂交，然后用荧光扫描仪检测信号。结果 基因芯片能够用于细菌检测，克服探针具有广泛和灵敏的特点，但是交叉反应明显；寡核苷酸探针具有较高的特异性，但是灵敏度稍差。结论 cDNA探针和寡核苷酸探针结合或设计几个不同的探针来指向同一株细菌很可能是将来基因芯片的检测方向。     

磷霉素氨丁三醇对尿标本中肠杆菌科细菌体外抗菌活性研究

目的了解磷霉素氨丁三醇对尿标本中肠杆菌科细菌的体外抗菌活性,指导临床合理用药。方法采用ATB系统对中段尿标本分离的1185株肠杆菌科细菌进行鉴定,纸片扩散法进行药敏试验和ESBI．S的检测,结果按CLSI2012年版标准判断;应用WHONET5．6软件分析病原菌的分布及耐药情况。结果1185株肠杆菌科细菌对磷霉素氨丁三醇的敏感率为90．8％,其中大肠埃希菌929株（78．4％）、肺炎克雷伯菌124株（10．5％）和奇异变形杆菌69株（5．8％）对磷霉素氨丁三醇的敏感率分别为93．1％、88．7％和79．7％。住院患者尿标本分离的肠杆菌科细菌对磷霉素氨丁三醇的敏感率低于门诊患者（87．0％对92．7％）。大肠埃希菌、肺炎克雷伯菌和奇异变形杆菌ESBLs的检出率分别为54．4％、38．7％和23．2％。产ESBLs的大肠埃希菌和肺炎克雷伯菌对磷霉素氨丁三醇的敏感率分别为89．5％和87．5％。结论尿标本培养获得的肠杆菌科细菌以大肠埃希菌为主,磷霉素氨丁三醇对尿标本分离的产ESBLs的大肠埃希菌和肺炎克雷伯菌的肠杆细菌科细菌具有良好的体外抗菌活性,可作为临床经验用药之一。     

老年细菌性肺炎痰标本的临床应用价值:基于16S Rdna测序方法

目的 采用16S rDNA高通量测序技术,比较不同采集方式获得的老年细菌性肺炎患者呼吸道标本的共有或独有细菌,评估质量合格的呼吸道标本的临床价值.方法 选取该院收治的10例诊断为细菌性肺炎且未经治疗的老年患者作为研究对象,分别采集自然咳痰(SH组)、纤维支气管镜吸痰(XT组)和纤维支气管镜肺泡灌洗液(XC组)标本,采用16 S rDNA高通量测序技术检测并比较3组标本细菌组成的相似性和差异性.结果 3组标本共有菌群959个属,3组标本独有细菌的属比较,差异有统计学意义(P<0.05),但非度量多维尺度分析显示,3组标本在属水平上总体细菌结构差异不明显.结论 16S rDNA高通量测序SH组标本细菌更丰富,但质量合格的3组标本在属水平上总体细菌结构差异不明显,SH组标本仍有一定的临床价值.     

急性淋巴细胞白血病患者血液中草螺菌的鉴定

目的 分离鉴定源于急性淋巴细胞白血病（ALL）患者血液中的未知细菌.方法 采用常规及VITEK32微生物GNI鉴定卡鉴定细菌,K-B法进行药敏试验,PCR法扩增细菌的16S rRNA基因,通过测序并与GenBank中相关序列进行比对.结果 常规生化反应和微生物自动化鉴定系统不能鉴定.16S rRNA PCR扩增产物经测序与GenBank中序列比对,发现与草螺菌属有99%同源性.结论 来源于ALL患者血液中的未知细菌是草螺菌.     

胆管结石患者Oddi括约肌中胆囊收缩素受体和一氧化氮合酶及其血中胆囊收缩素和一氧化氮水平的意义

目的探讨胆管结石患者Oddi括约肌中胆囊收缩素（CCK）受体和一氧化氮合酶（NOS）及其血清中CCK和NO含量的改变及意义。方法测定41例胆管结石患者和6例对照组血中的CCK和NO水平及Oddi括约肌中CCK受体和NOS含量。结果胆管结石组血中CCK含量[（38．91±4．85）pmol／L]、NO含量[（40．84±4．74）pmol／L]较对照组[（30．67±1．81）pmol／L]和[（32．81±1．11）pmol／L]明显升高;Oddi括约肌中CCK受体[（67．59±5．87）ng／L]及NOS含量[（457．52±45．40）ng／L]明显低于对照组[（78．99±1．71）ng／L]与[（519．61±11．38）ng／L];血中CCK、NO水平及Oddi括约肌中CCK受体、NOS含量在原发性肝内胆管结石组、胆囊结石伴胆管结石组、原发性胆总管结石组中有所不同。结论胆管结石患者Oddi括约肌中CCK受体及NOS含量下降导致Oddi括约肌功能下降,进而胆汁淤积,促进胆管结石的形成。     

Effects of bile salt and phospholipid hydrophobicity on lithogenicity of human gallbladder bile

Abstract Increased biliary bile salt and phospholipid hydrophobicity may promote nucleation of cholesterol crystals and gallstone formation. We therefore compared bile salt composition (determined by gas-liquid chromatography) in patients with cholesterol ( n = 35) and pigment ( n = 16) gallstones (group A). Bile salt composition and cumulative bile salt hydrophobicity index were not different between both stone types. Hydrophobicity index or % of individual bile salts did not correlate with cholesterol saturation index or nucleation time. In an additional 21 cholesterol stone patients (group B) biliary bile salt and phospholipid hydrophobicity as determined by high-pressure liquid chromatography did not correlate with cholesterol saturation index or nucleation time. In both group A and group B, cholesterol stone patients with cholesterol crystals in their fresh biles had a higher % deoxycholic acid, a lower % cholic acid and a higher bile salt hydrophobicity index than crystal-negative patients. This study indicates the need for further research on the role of bile salt hydrophobicity in the pathogenesis of gallstones.     

Gallbladder motility and cholesterol crystallization in bile from patients with pigment and cholesterol gallstones

BACKGROUND: Little is known about gallbladder motility in patients with black pigment stones when compared to cholesterol gallstone patients, or about their relationship to biliary composition, crystallization and stone characteristics. DESIGN: Fasting and postprandial gallbladder volumes were studied by ultrasonography in 49 gallstone patients with pigment (n = 14) or cholesterol (n = 35) stones and 30 healthy controls. After cholecystectomy stone composition, gallbladder wall inflammation, cholesterol saturation index and appearance of platelike cholesterol crystals in bile were evaluated in gallstone patients. RESULTS: Fasting gallbladder volume was significantly (P < 0.05) increased in cholesterol stone patients (31.7 +/- 1.9 mL) but not in pigment stone patients (21.9 +/- 3.1 mL), compared to controls (21.0 +/- 1.5 mL). Postprandial emptying was delayed in patients (half-emptying time: 31 +/- 2 min, 35 +/- 3 min, 24 +/- 2 min in cholesterol stone patients, pigment stone patients and controls, respectively, P < 0.05) and incomplete (residual volume: 43.2 +/- 2.7%, 40.0 +/- 4.3%, 15.8 +/- 1.6% min in cholesterol stone patients, pigment stone patients and controls, respectively, P < 0.05). The inflammation of the gallbladder wall was mild or absent in all cases. Biliary cholesterol saturation index was 152.3 +/- 8.5% and 92.9 +/- 4.8% in patients with cholesterol and pigment stones, respectively (P < 0.01). Whereas cholesterol crystals never appeared during 21 days in biles from patients with pigment stones, crystal observation time in patients with cholesterol gallstone was 5 days (median) and was significantly shorter in patients with multiple (4 days) than in patients with solitary (12 days) cholesterol stones (P = 0.0019). CONCLUSIONS: Patients with black pigment stones who do not have excess cholesterol and do not grow cholesterol crystals in bile have decreased gallbladder emptying, although to a lesser extent than patients with cholesterol stones. Thus, gallbladder stasis is likely to put a subset of subjects at risk for the formation of pigment gallstones, and pathogenic mechanisms need to be further investigated.     

Quantification of cholesterol nucleation promoting activity in human gallbladder bile

We have developed a simple method to quantitate cholesterol nucleation promoting activity in bile. The method makes use of the fact that gallbladder bile of cholesterol gallstone patients contains potent nucleation promoting activity. Gallbladder bile samples were serially diluted, routinely from 1/25 to 1/6,400. The diluted samples were mixed with a supersaturated model bile and the nucleation time (NT) of the mixtures was determined. The greatest dilution that resulted in a significant shortening of the NT was called the nucleation promoting activity titre (NPAT). The determination is independent of the original lipid content of the bile sample. The NPAT was measured in 14 gallbladder bile samples derived from patients with cholesterol gallstones and 9 controls. In all samples promoting activity was found. In the samples from the stone patients the NPAT was significantly elevated as compared to the patients without cholesterol stones (p = 0.01). Our results suggest that the cholesterol saturation index and the activity of cholesterol nucleation promoting factors are the most important factors in the pathogenesis of cholesterol gallstone disease. Assessment of the NPAT allows the differentiation of groups of patients with a normal cholesterol saturation index who are at risk for gallstone formation due to a high NPAT.     

腹腔镜联合十二指肠镜治疗胆囊结石合并胆总管结石

目的:探讨腹腔镜联合十二指肠镜治疗胆囊结石合并胆总管结石的临床疗效.方法:回顾分析2009年1月-2010年12月腹腔镜胆囊切除术(LC)联合十二指肠镜治疗35例胆囊结石合并胆总管结石患者的临床资料.所有患者均采取经十二指肠镜逆行胰胆管造影(ERCP),内镜下十二指肠乳头括约肌切开术(EST),取出胆总管结石,放置胆道塑料支架引流(ERBD).ERCP后3d内行LC,术后4d出院.出院后1～2周内再次行十二指肠镜取出胆道支架并行ERCP了解胆管有无残余结石.结果:35例患者均1次取净胆总管结石,1例EST术中出血,34例成功行LC,1例中转开腹行胆囊切除术.术后并发急性胰腺炎2例,所有患者均无胆漏、十二指肠穿孔、黄疸等并发症.结论:腹腔镜联合十二指肠镜治疗胆囊结石合并胆总管结石具有疗效确切、创伤小、恢复快等优点.     

经腹腔镜胆囊切除术选择性术中胆道造影的临床应用价值

目的探讨术中胆道造影在腹腔镜胆囊切除术（LC）中的临床应用价值。方法分析2006年9月至2010年12月北京丰台长辛店医院及东城区第一人民医院180例慢性结石性胆囊炎、急性结石性胆囊炎、胆源性胰腺炎患者行LC患者的临床资料,统计分析其中20患者在术中经胆囊管插管行胆管造影（IOC）的临床数据。结果 20例LC术中胆管造影18例成功,成功率90.0%,术中发现胆总管结石2例,经中转开腹行胆总管切开取石,胆管变异1例;平均造影时间15±10.50 min。结论 LC中胆管造影操作较为简单方便、显影清晰、成功率高,既能发现术前未能发现的胆总管结石,又能发现胆管解剖变异;并能够有效地降低胆管残石率,及时发现术中胆管损伤,提高了LC的手术质量和安全性。     

Cholesterol gallstone pathogenesis: a study of potential nucleating agents for cholesterol crystal formation in bile

Cholesterol monohydrate crystal formation was measured quantitatively in model bile solutions, which were supersaturated with cholesterol, by a radiochemical method and qualitatively in human gallbladder bile by polarizing microscopy. Various agents, which have been postulated to act as nucleating factors for cholesterol crystal and gallstone formation, were added to bile and their effect on the appearance of cholesterol crystals was determined. These agents included calcium salts found in gallstones (calcite, aragonite, apatite, bilirubinate), Escherichia coli bacteria, pigment residues from cholesterol gallstones, bilirubin and several mucin preparations. Human gallbladder bile, which was collected from patients with and without cholesterol gallstones, was also mixed with model bile to examine whether nucleating or anti-nucleating factors were present. None of the agents tested markedly and consistently promoted cholesterol monohydrate crystal formation in model or human bile, except seed crystals of cholesterol monohydrate which were used as a control. Human gallbladder bile from obese patients without gallstones delayed the appearance of cholesterol crystals in model bile solutions, whereas gallbladder bile from gallstone patients did not. These results do not provide experimental support for the hypothesis that calcium salts and pigment material found in gallstones, or gallbladder mucin at concentrations less than 10 mg/ml, act as nucleating agents for cholesterol crystal and stone formation. The difference between gallbladder biles from patients with and without gallstones in their propensity to form cholesterol crystals may be due to the presence of an anti-nucleating factor in normal bile.     

Biliary lipid composition in patients with cholesterol and pigment gallstones and gallstone-free subjects: deoxycholic acid does not contribute to formation of cholesterol gallstones

BACKGROUND: Four main disturbances have been attributed to cholesterol gallstone disease: hypersecretion of cholesterol from the liver with cholesterol supersaturation in bile; disturbed motility with defective absorption and secretion by the gallbladder; increased crystallisation of cholesterol in the gallbladder bile; and slow intestinal transit with increased amount of deoxycholic acid in the bile acid pool. We aimed to evaluate the biliary lipid composition in a large series of gallstone patients, with emphasis on the amount of deoxycholic acid and with respect to number of stones, compared to gallstone free subjects. MATERIALS AND METHODS: Bile was sampled during operations through puncture of the gallbladder from 145 cholesterol gallstone patients, 23 patients with pigment stones and 87 gallstone free patients undergoing cholecystectomy. Biliary lipid composition, cholesterol saturation, bile acid composition, nucleation time and cholesterol crystals were analysed. RESULTS: The patients with cholesterol gallstones showed higher molar percentage of cholesterol, lower total biliary lipid concentration, higher cholesterol saturation, shorter nucleation time and higher proportion of crystals in bile than the other groups. The nucleation time was significantly shorter in multiple cholesterol gallstone patients, but this was not due to higher cholesterol saturation. Male cholesterol gallstone patients showed higher cholesterol levels, lower total biliary lipid concentration, and higher cholesterol saturation in bile than female patients. There was no difference in biliary content of deoxycholic acid, but significantly lower content of cholic acid in gallstone patients compared to gallstone free patients. CONCLUSIONS: We conclude that deoxycholic acid does not contribute to gallstone formation in cholesterol gallstone patients. The short nucleation time in patients with multiple cholesterol stones is not due to higher cholesterol saturation.