胰胆管合流异常患者胆汁对人胆管癌细胞生长的影响

目的　研究胰胆管合流异常患者 (APBDU)胆汁对人胆管癌细胞QBC93 9生长的影响。方法　应用四唑氮蓝比色 (MTT)法检测QBC93 9增殖状况 ,应用流式细胞仪测定细胞周期和凋亡 ,采用酶联免疫吸附试验 (ELISA)检测前列腺素E2 (PGE2 )含量。结果　APBDU胆汁与正常对照组胆汁比较明显促进QBC93 9细胞增殖 (P <0 .0 5 ) ,这种促癌活性可被环氧合酶 2 (COX 2 )选择性抑制剂Celecoxib拮抗。用APBDU患者胆汁处理 2 4h的QBC93 9细胞增殖指数显著上升(P <0 .0 5 ) ,S期细胞比例为 ( 3 2 .2 4± 1.3 3 ) % ,比正常对照组 ( 10 .5 6± 1.47) %明显增高 (P <0 .0 5 ) ,G0 /G1期细胞比例为 ( 4 9.72± 4.13 ) % ,比正常对照组 ( 74.81± 5 .10 ) %明显降低 (P <0 .0 5 )。结论　APBDU胆汁具有潜在的促癌活性 ,这种促癌活性依赖COX 2和PGE2 途径。     

胆管结石患者胆汁对人胆管癌细胞增殖的影响

目的观察胆管结石患者胆汁对人胆管癌细胞QBC939生长的影响 ,探讨胆管结石与胆管癌发生、发展的关系。方法应用噻唑蓝比色法检测 2 0份胆管结石患者胆汁和 10份正常胆汁对QBC939增殖的影响 ,应用流式细胞仪测定细胞周期和凋亡。结果胆管结石患者胆汁与正常胆汁比较 ,明显促进QBC939细胞增殖 ,用胆管结石患者胆汁处理 4 8h的QBC939细胞增殖指数显著上升 (P <0 0 1) ,胆管结石胆汁组 (47%± 10 % )S期细胞比例比正常胆汁组 (2 3%± 3% )明显增高 (P <0 0 1) ,G0 /G1期细胞比例 (42 %± 8% )比正常胆汁组 (6 3%± 10 % )明显降低 (P <0 0 5 )。结论胆管结石患者胆汁具有潜在的促增殖活性 ,胆管结石与胆管癌的发生、发展关系密切     

胰胆管合流异常患者胆汁对人胆管癌细胞生长的影响

目的　研究胰胆管合流异常患者胆汁对人胆管癌细胞生长的影响 ,探讨胰胆管合流异常与胆管癌发生和发展的关系。方法　应用四唑氮蓝 (MTT)比色法检测胰胆管合流异常患者胆汁对人胆管癌细胞 QBC939增殖的影响 ,应用流式细胞仪 (FCM)测定细胞周期和细胞凋亡 ,采用酶联免疫吸附试验 (EL ISA)检测前列腺素 E2 (PGE2 )比例。结果　胰胆管合流异常患者胆汁与正常对照组胆汁比较明显促进胆管癌细胞 QBC939的增殖 (P<0 .0 1) ,这种促癌活性可被 COX- 2选择性抑制剂 celecoxib拮抗。细胞周期分析用胰胆管合流异常患者胆汁处理 2 4 h的 QBC939细胞增殖指数显著上升 (P<0 .0 1) ,S期细胞比例为 32 .2 4± 1.33% ,比正常对照组 (10 .5 6± 1.4 7% )明显增高 (P<0 .0 5 ) ,G0 / G1期细胞比例为 4 9.72 %± 4 .13% ,比正常对照组 (74 .81± 5 .10 % )明显降低 (P<0 .0 4 2 ) ,但胰胆管合流异常患者胆汁对 QBC939细胞凋亡率无明显影响 (P=0 .882 )。结论　胰胆管合流异常患者胆汁具有潜在的促癌活性 ,这种促癌活性依赖 COX- 2和 PGE2 途径     

PTTG对胆管癌细胞生长和5-FU敏感性影响的研究

目的探讨抑制垂体瘤转化基因(PTTG)表达后胆管癌细胞体外生长的变化及其对5-FU化疗敏感性的影响。方法将反义PTTG真核表达载体pcDNA3.1-PTTGas和空白载体pcDNA3.1(+)转染入胆管癌细胞株QBC939中,筛选获得稳定表达株tQBC939(PTTG-)和tQBC939(pcDNA3.1),并设立未转染的QBC939细胞对照组,绘制细胞生长曲线,以MTT法检测细胞增殖力的变化,流式细胞仪检测细胞周期的比例变化,并采用5-FU处理细胞后,以MTT检测3组细胞的生长抑制率,计算IC50值,流式细胞仪和Hoechst染色法检测各组细胞凋亡情况。结果 tQBC939(PTTG-)组与tQBC939(pcD-NA3.1)组和QBC939组比较,前者生长较为迅速,S期细胞比例增多,G2/M期细胞比例减少(P0.05),增殖指数较高(P0.05);经5-FU处理后,tQBC939(PTTG-)的IC50值明显低于tQBC939(pcD-NA3.1)和QBC939细胞(P0.05),细胞凋亡增多。结论转染PTTG反义DNA可增强胆管癌细胞的增殖力和对5-FU化疗的敏感性。     

幽门螺杆菌感染的成石胆汁促进人胆管癌细胞增殖的研究

目的探讨幽门螺杆菌感染的成石胆汁对人胆管癌细胞QBC939生长的影响，阐明幽门螺杆菌感染与胆管癌发生、发展的内在关系。方法应用噻唑蓝比色法检测20份幽门螺杆菌阳性胆管结石患者胆汁、20份幽门螺杆菌阴性胆管结石患者胆汁以及20份正常胆汁对QBC939细胞增殖的影响，运用流式细胞仪测定细胞周期与凋亡。结果与幽门螺杆菌阴性胆管结石患者胆汁和正常胆汁比较，幽门螺杆菌感染的成石胆汁明显促进QBC939细胞增殖（P〈0.05），用幽门螺杆菌感染的成石胆汁处理48h的QBC939细胞增殖指数显著上升（P〈0.01），S期细胞比例明显增高（P〈0.05），G0／G1期细胞比例明显降低（P〈0．05）。结论幽门螺杆菌感染的成石胆汁具有强大的潜在促增殖活性，胆道幽门螺杆菌感染与胆管癌的发生、发展关系密切。     

雷帕霉素对人骨肉瘤细胞增殖和凋亡的影响

目的 观察雷帕霉素(RAPA)对人骨肉瘤细胞U-2OS和Saos-2增殖和凋亡的影响并探讨其机制.方法 体外培养骨肉瘤细胞株U-2OS和Saos-2;噻唑蓝(MTT)比色法和流式细胞仪检测不同浓度雷帕霉素对骨肉瘤细胞增殖和凋亡的影响;Western blot检测雷帕霉素作用后骨肉瘤细胞Fas蛋白表达的变化.结果 MTT检测显示雷帕霉素可抑制入骨肉瘤细胞增殖,在雷帕霉素浓度达到20 nmol/L时,抑制作用显著提高(P＜0.05),增殖抑制率分别达到31.2％和28.7％.流式细胞检测显示在雷帕霉素浓度达到20 nmol/L时,其诱导骨肉瘤细胞凋亡的作用显著增强(P＜0.05).Western blot检测显示经雷帕霉素作用48 h后的骨肉瘤细胞Fas蛋白的表达明显增强.结论 雷帕霉素对入骨肉瘤细胞株U-2OS和Saos-2的增殖有抑制作用并可诱导其发生凋亡,其可能是通过上调Fas蛋白的表达而发挥作用的.     

羧甲基壳聚糖抗氧化损伤诱导雪旺细胞凋亡的作用及机制研究

[目的]探讨羧甲基壳聚糖(carboxymethylated chitosan,CMCS)对氧化应激诱导雪旺细胞(schwann cells,SCs)凋亡的保护作用及作用机制。[方法]体外培养SCs,S-100免疫荧光染色鉴定。将SCs分为空白对照组、H2O2诱导组、H2O2加CMCS处理组。通过CCK-8检测不同组SCs增殖情况,流式细胞仪计数检测SCs早期凋亡率,检测各组细胞内超氧化物歧化酶(SOD)及丙二醛(MDA)的含量,Real-time PCR技术检测Bcl-2及Bax mRNA表达水平,Western blot法检测Bcl-2,Bax,Caspase-3、-9的表达水平。[结果]H2O2诱导SCs后其细胞增殖活性明显降低,加入不同浓度CMCS后增殖活性增加。流式细胞技术检测结果表明,经H2O2诱导SCs早期凋亡率增加,加入50²00μg/ml CMCS后凋亡率逐渐降低。经H2O2诱导组SCs与空白对照组比较SOD含量明显减少,MDA含量明显增加(P<0.05);加入CMCS后SOD含量增加,MDA含量减少(P<0.05)。Real-time PCR及Western blot检测结果表明经H2O2诱导SCs内Bcl-2表达降低,Bax,Caspase-3、-9表达增高,加入CMCS后Bcl-2表达恢复,Bax,Caspase-3、9表达抑制。[结论]CMCS对H2O2诱导SCs凋亡具有保护作用。     

血管生长抑素基因联合生长抑素治疗人胰腺癌的体外研究

目的 探讨血管生长抑素基因联合生长抑素在体外对人胰腺癌细胞BXPC-3和血管内皮细胞ECV-304增殖的抑制作用.方法 重组pcDNA3/angio质粒转染人胰腺癌细胞BXPC-3,利用RT-PCR和Western blot检测其表达及分泌血管生长抑素的情况;MTT法和流式细胞仪检测生长抑素和血管生长抑素对BXPC-3和ECV-304细胞增殖的影响.结果 转染后的BXPC-3细胞表达并分泌血管生长抑素.一定浓度(≥10μg/ml)的生长抑素对BXPC-3细胞的增殖有明显抑制作用(P<0.01),并在一定浓度范围内呈剂量依赖性,同时能诱导BXPC-3细胞凋亡(P<0.01);但其对ECV-304细胞的增殖无明显影响(P>0.05).血管生长抑素对ECV-304细胞的增殖有明显抑制作用(P<0.01),并能诱导其凋亡(P<0.01);但对BXPC-3细胞的增殖无明显影响(P>0.05).血管生长抑素联合生长抑素对BXPC-3及ECV-304细胞的增殖均有明显抑制作用(P<0.01),也可诱导其凋亡(P<0.01),但无协同增强效应.结论 ①生长抑素主要通过直接抑制胰腺癌细胞增殖、促进其凋亡而发挥抗胰腺癌作用;在体外其对血管内皮细胞无抑制作用.②血管生长抑素主要通过特异性地抑制胰腺癌血管内皮细胞的增殖并诱导其凋亡,从而抑制新生毛细血管的生成,致胰腺癌细胞坏死和肿瘤消退.     

抑制miRNA-21表达对胆管癌细胞凋亡、侵袭的影响及其靶基因探讨

目的 探讨抑制miRNA-21表达对胆管癌细胞株凋亡、侵袭的影响及其可能调控的靶基因。方法通过RNAi技术抑制胆管癌细胞株RBE中miRNA-21的表达并加以验证。通过荧光定量PCR技术及蛋白质免疫印迹法(Western blot)检测RBE细胞中RECK基因表达的改变。应用流式细胞仪检测细胞凋亡的变化。用体外侵袭实验分析胆管癌细胞侵袭能力的改变。结果miRNA-21在胆管癌细胞株RBE中的表达得到有效抑制,并且RECK基因及其蛋白的表达明显上调。流式细胞仪显示RBE细胞凋亡率明显增高,且抑制miRNA-21的表达后胆管癌细胞的侵袭性明显下降。结论抑制miRNA-21的表达可促进RBE细胞的凋亡,并降低其侵袭能力,RECK作为miRNA-21的靶基因参与调控过程。     

阻断Hedgehog信号通路可抑制胆管癌细胞QBC939的生长

目的 研究Hedgehog信号通路特异性阻断剂环靶明(cyclopamine)对人胆管癌细胞系QBC939增殖与凋亡的影响.方法 用MTT比色法检测环靶明对QBC939细胞增殖的抑制作用,流式细胞术检测细胞凋亡率.RT-PCR法分别检测环靶明处理前后PTCH1、GLI1、EGFR在QBC939中的mRNA表达及变化.Western blot检测环靶明处理前后PTCH1、GLI1、EGFR在QBC939中的蛋白表达及变化.结果 环靶明抑制QBC939细胞的增殖,其作用呈剂量和时间依赖性.经5、10、20 μmol/L的环靶明作用48 h后,QBC939细胞的凋亡率逐渐升高,明显高于对照组的凋亡率(P＜0.01).PTCH1、GLI1、EGFR的mRNA和蛋白均在QBC939中表达,环靶明下调QBC939的PTCH1、GLI1、EGFR表达.结论 阻断Hedgehog信号通路能抑制胆管癌细胞QBC939的增殖,促进其凋亡.     

胆道再次手术235例分析

目的总结胆道再次手术的经验，探讨胆道再次手术的原因、临床特点及处理方法。方法对1996年7月至2005年6月收治的235例胆道再次手术的临床资料进行回顾性分析。结果胆道再次手术的主要原因是肝胆管结石残留或复发，占全组病例的82．2％；其次为胆肠吻合术后狭窄，胆管囊肿，胆管狭窄及胆管肿瘤。再次手术治疗方式以肝叶切除（占66％）合并胆肠吻合或T管引流为主。胆道再次手术后并发症发生率为25％。结论胆道再次手术既有胆道疾病本身因素，亦有医源性因素；充分的术前准备，术中仔细探查，选择合理手术方式及合适的术后辅助治疗是减少胆道再次手术的关键。     

Bile duct cysts

Bile duct cysts are uncommon lesions that are found in adult and pediatric patients. Current concepts regarding epidemiology, etiology, classification, clinical diagnosis, and surgical treatment are reviewed. Bile duct cysts are associated with abnormal junctional anatomy of the pancreatic and bile ducts and with biliary tract cancer. When possible, complete cyst excision is the recommended treatment.     

Bile duct adenomas with endocrine component. Immunohistochemical study and comparison with conventional bile duct adenomas.

Bile duct adenomas are small nodules that are usually found incidentally on the liver surface at abdominal surgery or autopsy. We recently analyzed two such lesions that, in addition to the typical small caliber ducts, contained periductular nests and clusters of uniform round cells, suggestive of endocrine cell proliferation. Follow-up of these patients did not show endocrine tumors elsewhere. The lesions were studied by immunohistochemistry (avidin-biotin-peroxidase technique) and compared with conventional bile duct adenomas (seven cases). The results showed these cells to decorate with several endocrine markers, namely, neuron-specific enolase, chromogranin, synaptophysin, and Leu-7. Endocrine markers were not seen in the cells of conventional bile duct adenomas. Epithelial markers, that is, cytokeratin (CAM 5.2 antibody) and epithelial membrane antigen, were expressed by the cells composing both conventional bile duct adenomas and those with endocrine-like cells, although with less intensity in the endocrine cell clusters. We suggest that some bile duct adenomas contain endocrine cell proliferations that morphologically may resemble a small carcinoid tumor or the so-called pulmonary tumorlet. Neurosecretory granules have previously been identified in some cholangiocarcinomas and in bile duct proliferation associated with cholestasis. The endocrine clusters in biliary adenomas may constitute a diagnostic pitfall and must be separated from metastases of carcinoids or islet cell tumors.     

腹腔镜胆囊切除术中胆管损伤防治分析

目的探讨腹腔镜胆囊切除术（laparoscopic cholecystectomy,LC）术中胆管损伤原因,总结处理经验。方法回顾性分析1998年10月-2006年8月1 528例LC术中9例（0.59%）胆管损伤的资料。结果本组7例术中发现胆管损伤,4例予局部压迫处理,1例术中置入T管引流,2例中转开腹手术。另有2例术后出现胆汁性腹膜炎而二次手术,术中发现胆总管损伤,予T管支撑引流治愈。开展LC的前500例中发生胆管损伤6例（1.20%）,其中中转开腹手术2例（0.40%）;500例以后的LC,胆管损伤率为0.29%（3/1 028）,中转开腹率为0。结论LC术中胆管损伤多发生在复杂病理改变的病例,强化对解剖变异、高危病例及高危技术的认识是避免胆管损伤的关键;一旦发生LC术中不能良好处理的胆管损伤,及时中转开腹手术是理性的选择。     

Small Bile Duct Reconstruction of the Caudate Lobe in Living-Related Liver Transplantation

OBJECTIVE: To justify the technique of biliary reconstruction without mucosa-to-mucosa alignment for reconstructing the caudate lobe bile duct. SUMMARY BACKGROUND DATA: The use of a left hepatic lobe graft with the caudate lobe (LHGC) has been introduced to resolve the problem of small-for-size grafts in living-related liver transplantation. The authors have found that the LHGCs occasionally have independent openings of the bile duct of the caudate lobe. METHODS: The graft bile ducts were anastomosed to Roux-en-Y jejunal loops. The main left hepatic ducts were reconstructed in a standard manner. Small bile ducts of the caudate lobe were anastomosed to the intestine without mucosa-to-mucosa alignment, with an external biliary drainage tube, positioned transanastomotically. RESULTS: In 8 of the 19 patients who received LHGCs, nine bile ducts of the caudate lobe (median 1 mm) were reconstructed. After surgery there was no bile leakage. In five of the eight patients, the tubes were removed a median of 92 days after transplantation. Bile duct dilatation had not been observed at a median of 363 days after surgery. CONCLUSIONS: The authors consider their technique of biliary reconstruction without mucosa-to-mucosa alignment useful for the safe reconstruction of small bile ducts of the caudate lobe.     

Bile leak test by indocyanine green fluorescence images after hepatectomy

Bile leak remains a serious complication after hepatectomy. The conventional leak test by intrabiliary injection of normal saline solution is not sensitive. The authors present a new bile leak test using indocyanine green (ICG) fluorescence. After hepatic transection, ICG solution (.05 mg/mL) was intrabiliarily injected through a transcystic tube under distal common bile duct clamping, and fluorescent images were visualized using an infrared camera system. The ICG leak test was performed in 27 patients undergoing hepatectomy without biliary reconstruction. Bile leaks were intraoperatively found in 8 patients and fixed, resulting in no postoperative leaks. There was no adverse reaction to ICG. In contrast, postoperative bile leaks occurred in 2 of 32 patients who received the conventional leak test with normal saline solution between April 2007 and March 2008. The new bile leak test by ICG fluorography is useful to prevent postoperative bile leak.     

带蒂脐静脉瓣胆道修复术与远期疗效

目的　总结带蒂脐静脉瓣胆道重建的临床经验和远期随访结果。方法　第一种术式 ,胆管上下端后壁对端吻合 ,前壁纵行切开 ,其缺损以带蒂脐静脉瓣修复 2 2例 ;第二种术式 ,胆管狭窄切开、整形 ,胆管缺损以带蒂脐静脉瓣修复 19例。结果　本组无死亡病例 ,术后达 7年以上 2 5例 ,其中再手术 3例 ,术后 7- 12年ERCP 10例 ,MRCP和双剂量静脉胆道造影各 1例 ,胆道显影均正常 ;轻度胆管炎 2例 ;另 8例无任何胆道感染症状 ,B超胆道未见异常。结论　带蒂脐静脉瓣胆道重建是一种理想的胆道修复手术。     

Altered Bile Composition in the Gallbladder and Common Bile Duct of Patients with Anomalous Pancreaticobiliary Ductal Junction

The high incidence of biliary tract carcinoma in patients with anomalous pancreaticobiliary ductal junction (APBDJ) has been well documented. Elevation of the secondary and free bile acid (FBA) concentrations is considered a risk factor for biliary carcinogenesis in these patients. Bile from the gallbladder and common bile duct in 12 patients with APBDJ was analyzed and compared with gallbladder bile from 19 patients with gastric cancer and a normal hepatobiliary tract. The concentrations of secondary bile acids were significantly lower in the APBDJ group than in the control group, and FBA concentrations were not detected in the gallbladder in either group. The lysolecithin (LL) in the phospholipid, which is produced from lecithin by activated phospholipase A₂ in refluxing pancreatic juice, was significantly elevated in the APBDJ group. Elevation of the LL concentration in the bile is one of the factors for the development of biliary tract carcinoma in patients with APBDJ.     

Bile acid receptors in colorectal cancer

Bile acids are thought to be involved in both the aetiology and development of colorectal cancer. In this study the existence of specific bile acid receptor proteins has been postulated. A receptor assay which involved labelling with 14C-deoxycholic acid was performed as well as autoradiography using 3H-deoxycholic acid. In an initial study resected colorectal cancer and adjacent histologically normal colorectal mucosa from 39 patients were studied, as were samples of normal gastric mucosa, cancers and benign colorectal tumours. Specific receptors to deoxycholic acid were detected in 12 (30.8 per cent) of the colorectal cancers, but in only 1 (2.6 per cent) of the samples from normal colorectal mucosa (X2 = 11.16, P less than 0.005). No deoxycholic acid receptors were detected in any other tissue studied. Autoradiographs of colorectal cancers showed binding of 3H-deoxycholic acid in receptor-positive tumour tissue. These findings might provide some explanation for the evidence linking bile acids with the disease.     

Preservation of Bile Ductules Mitigates Bile Duct Loss

The finer branches of the biliary tree (FBBT) contain a regenerative compartment. We hypothesized that preservation of the FBBT together with its microvasculature will lead to recovery of biliary damage and prolonged preservation of bile ductules during the development of chronic liver allograft rejection. The interlobular bile ducts, portal bile ductules and extraportal biliary cells with and without microvessels were studied in sequential biopsies in five patients who fulfilled the Banff criteria of early chronic rejection (CR) (imminence group). Biopsies of CR patients (n = 12) served as controls. Biopsies were double immunostained with CD34 (microvessels) and cytokeratin 7 (biliary structures). Proliferation and proangiogenic activity were assessed with Ki67 and VEGF-A immunostaining. Severe damage of bile ducts in the imminence group did not progress to significant bile duct loss. This was associated with a high proliferative activity in all biliary structures and preservation of the microvascular compartment. VEGF-A expression was increased in all but the reperfusion biopsies. In conclusion, both regenerative activity of the FBBT and an intact microvascular compartment are associated with less damage of the biliary tree and could therefore be prerequisites for biliary regeneration.