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The objective of this study was to carry out a meta-analysis of the efficacy of gemcitabine+platinum agent regimens in the treatment of advanced biliary tract cancer (BTC). PubMed and Google Scholar were searched using the following combination of search terms: gemcitabine, oxaliplatin, cholangiocarcinoma, biliary, gallbladder, bile duct. Studies were eligible for inclusion in the meta-analysis if they were randomized trials on the use of gemcitabine plus a platinum agent for the treatment of advanced (unresectable or metastatic cancer) BTC. Outcomes of interest were response rate, overall survival, and progression-free survival. Pooled odds ratios/differences in median survival and 95% confidence intervals (CIs) were determined for each outcome. A total of 47 records were identified in the initial search. Ultimately, three open-label randomized trials (two phase 2 and one phase 3) met the eligibility criteria and were included in the meta-analysis. Two studies compared gemcitabine plus cisplatin with gemcitabine alone, whereas the other study compared gemcitabine plus oxaliplatin with fluorouracil-folinic acid. The total number of patients in the studies ranged from 54 to 410. The overall analyses revealed that all survival outcomes assessed were significantly more favorable for patients treated with gemcitabine plus platinum agents than for patients not treated with this combination. Response rates: odds ratio=2.639, 95% CI=1.210-5.757, Z=2.439, P=0.015; pooled difference in median overall survival=3.822 months, 95% CI=1.798-5.845 months, Z=3.702, P

Excision repair cross-complementary gene 1 (ERCC1) is a downstream regulatory target of fibroblast growth factor receptor 2 (FGFR2); however, the mechanism of its action has not been elucidated. The cascades downstream of FGFR2 include the PKC, Ras/Raf/MEK/ERK, JAK/STAT and PI3K pathways. ERCC1 is considered to be a closely related downstream target gene of extracellular signal-regulated kinase (ERK)1/2, since ERCC1 mRNA and protein levels may be inhibited by the ERK inhibitor U0126. It was hypothesized that FGFR2, which specifically binds with fibroblast growth factor 7 (FGF7), may regulate ERCC1 gene expression through the ERK signaling pathway. The aim of the present study was to explore the association between the regulatory effect of FGFR2 on ERCC1 gene expression and the p-ERK1/2 signaling pathway in a drug-resistant hepatocellular carcinoma (HCC) cell line. The drug-resistant cell line HepG2/OXA and its parental cell line HepG2 were transfected with Bek shRNA in the logarithmic growth phase. Transfected and untransfected HepG2 and HepG2/OXA cells were then stimulated with FGF7 and changes in the protein expression of FGFR2, p-ERK1/2 and ERCC1 was detected with western blot analysis. Following transfection, HepG2/T and HepG2/OXA/T cells were observed to grow stably in a screening medium containing puromycin. The western blot analysis demonstrated a significant decrease in the protein expressions of FGFR2, p-ERK1/2 and ERCC1 in HepG2/T and HepG2/OXA/T cells as compared to untransfected cells. Expression of FGFR2, p-ERK1/2 and ERCC1 in HepG2/OXA cells was significantly increased compared to the parental HepG2 cells. Following stimulation with FGF7, the expression of FGFR2, p-ERK1/2 and ERCC1 was increased, with significant differences between HepG2 and HepG2/OXA cells in the expression of p-ERK1/2 and ERCC1. No differences were detected in the protein levels following Bek shRNA transfection in HepG2/T and HepG2/OXA/T cells. In conclusion, the FGFR2-mediated ERK1/2 signaling pathway in HCC cells plays an important role in the regulation of ERCC1 expression.

Objective To clarify the relationship between loss of expression of DPC4 proteins and pathogenesis of biliary tract carcinoma.

To explore the improvement of typing and reasonable surgical treatment for pancreatic ductal stone (PDS).Totally 89 patients with pancreatic ductul stone treated underwent surgeries from January 2000 to December 2012 were involved into this study. There were 57 male and 32 female patients, the average age was (52 ± 23) years. According to the magnetic resonance cholangiopancreatography imaging and finding during surgery, pancreatolithiasis was classified into three types: type I, the stones were located in the main pancreatic duct; type II, the stones were located both in main and branch pancreatic duct; type III, the stones were diffusely scattered in the branch pancreatic duct; the position of PDS within pancreatic parenchyma were subtitled. In this group, 43 type I PDS were extracted with endoscopic papillotomy or endoscopic pancreatic sphincterotomy, or pancreatolithotomy plus pancreato-jejunal lateral anastomosis with wide anastomotic stoma; 39 type II cases were treated by pancreatolithotomy plus pancreato-jejunal lateral anastomosis or/and resection of pancreatic section; 7 type III PDS were managed with resection of pancreatic section.All surgeries were performed successfully. Among complications, 6 cases (6.7%) were pancreatic leakage which recovered after systematic non-surgical treatment, 2 cases (2.2%) were anastomotic bleeding which led to 1 death, 6 cases (6.7%) were residual pancreatolithiasis in branch pancreatic duct type. Seventy-eight patients were followed up for 6 to 131 months, 57 cases were still alive so far. Five cases were intermittent abdominal pain, 7 cases were diabetes resulted from 2 subtotal pancreatectomy and 5 distal pancreatectomy, 5 cases occurred pancreatolithiasis recurrence and 3 underwent secondary surgeries.The basis of this modified typing of pancreatolithiasis is the position of stone in pancreatic duct rather than pancreas parenchyma. It is more important and valuable for surgical principle of taking stones out completely and maintaining pancreatic function.

AIM: To investigate the molecular mechanisms underlying the reversal effect of emodin on platinum resistance in hepatocellular carcinoma. METHODS: After the addition of 10 μmol/L emodin to HepG2/oxaliplatin (OXA) cells, the inhibition rate (IR), 50% inhibitory concentration (IC50) and reversal index (IC50 in experimental group/IC50 in control group) were calculated. For HepG2, HepG2/OXA, HepG2/OXA/T, each cell line was divided into a control group, OXA group, OXA + fibroblast growth factor 7 (FGF7) group and OXA + emodin group, and the final concentrations of FGF7, emodin and OXA in each group were 5 ng/mL, 10 μg/mL and 10 μmol/L, respectively. Single-cell gel electrophoresis was conducted to detect DNA damage, and the fibroblast growth factor receptor 2 (FGFR2), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and excision repair cross-complementing gene 1 (ERCC1) protein expression levels in each group were examined by Western blotting. RESULTS: Compared with the IC50 of 120.78 μmol/L in HepG2/OXA cells, the IC50 decreased to 39.65 μmol/L after treatment with 10 μmol/L emodin; thus, the reversal index was 3.05. Compared with the control group, the tail length and Olive tail length in the OXA group, OXA + FGF7 group and OXA + emodin group were significantly increased, and the differences were statistically significant (P < 0.01). The tail length and Olive tail length were lower in the OXA + FGF7 group than in the OXA group, and this difference was also statistically significant. Compared with the OXA + FGF7 group, the tail extent, the Olive tail moment and the percentage of tail DNA were significantly increased in the OXA + emodin group, and these differences were statistically significant (P < 0.01). In comparison with its parental cell line HepG2, the HepG2/OXA cells demonstrated significantly increased FGFR2, p-ERK1/2 and ERCC1 expression levels, whereas the expression of all three molecules was significantly inhibited in HepG2/OXA/T cells, in which FGFR2 was silenced by FGFR2 shRNA. In the examined HepG2 cells, the FGFR2, p-ERK1/2 and ERCC1 expression levels demonstrated increasing trends in the OXA group and OXA + FGF7 group. Compared with the OXA group and OXA + FGF7 group, the FGFR2, p-ERK1/2, and ERCC1 expression levels were significantly lower in the OXA + emodin group, and these differences were statistically significant. In the HepG2/OXA/T cell line that was transfected with FGFR2 shRNA, the FGFR2, p-ERK1/2 and ERCC1 expression levels were significantly inhibited, but there were no significant differences in these expression levels among the OXA, OXA + FGF7 and OXA + emodin groups. CONCLUSION: Emodin markedly reversed OXA resistance by enhancing OXA DNA damage in HepG2/OXA cells, and the molecular mechanism was related to the inhibitory effect on ERCC1 expression being mediated by the FGFR2/ERK1/2 signaling pathway.

Background. None of treatment options for Cholangiocarcinoma (CCA), including surgery, adjuvant radiotherapy and chemotherapy, and ultimately liver transplantation, have been shown to substantially improve the survival rate in patients with CCA. Valproic acid (VPA), a histone deacetylase inhibitor, has been shown to display potent antitumor effects. In this study, sodium valproate, the clinically available form of VPA, was tested for its ability to inhibit the growth of cholangiocarcinoma cells, bothin vitroandin vivo. Materials and Methods.Cholangiocarcinoma cells (TFK-1, QBC939, and CCLP1) of different origins were treated with sodium valproate to determine their effects on cell proliferation and differentiation, cell cycle regulation, apoptosis, and autophagy. Thein vivoeffects of sodium valproate on cholangiocarcinoma growth were assessed using a xenograft mouse model injected with TFK-1 cells.Results. Sodium valproate inhibited cholangiocarcinoma cell growth by inducing cell cycle arrest, cell differentiation, and apoptosis; sodium valproate effects were independent of autophagy. Tumor growth inhibition was also observedin vivousing TFK-1 xenografts.Conclusion. Thein vitroandin vivooutcomes provide preclinical rationale for clinical evaluation of sodium valproate, alone or in combination with other drugs, to improve patient outcome in cholangiocarcinoma.

AIM: To investigate the reciprocal modulation between microRNA (miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpG-binding domain protein (MBD)2. METHODS: MiR-373 expression was examined using the TaqMan miRNA assay. Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction, and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay. Mutation analysis was conducted using the QuikChange™ Site-Directed Mutagenesis kit. The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region (3’UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay. RESULTS: In hilar cholangiocarcinoma, miR-373 decreased and was closely associated with poor cell differentiation, advanced clinical stage, and shorter survival. The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373. MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373. Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island, and miR-373 negatively regulated MBD2 expression through targeting the 3’UTR. CONCLUSION: MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

AIM: To identify methylation profile and novel tumor marker of extrahepatic cholangiocarcinoma (CCA) with high throughout microarray. METHODS: Differential methylation profile was compared between normal bile duct epithelial cell lines and CCA cell lines by methyl-DNA immunoprecipitation (MeDIP) microarray. Bisulfite-polymerase chain reaction (BSP) was performed to identify the methylated allels of target genes. Expression of target genes was investigated before and after the treatment with DNA demethylating agent. Expression of candidate genes was also evaluated by immunofluorescence in 30 specimens of CCA tissues and 9 normal bile duct tissues. RESULTS: Methylation profile of CCA was identified with MeDIP microarray in the respects of different gene functions and signaling pathways. Interestingly, 97 genes with hypermethylated CpG islands in the promoter region were homeobox genes. The top 5 hypermethylated homeobox genes validated by BSP were HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%). Expression of these genes was reactivated with 5’-aza-2’-deoxycytidine. Significant expression differences were found between normal bile duct and extrahepatic CCA tissues (66.67%-100% vs 3.33%-10%). CONCLUSION: HOXA2, HOXA5, HOXA11, HOXB4 and HOXD13 may work as differential epigenetic biomarkers between malignant and benign biliary tissues.

Background Hepatocellular carcinoma (HCC) is the third most common cause of cancer related mortality worldwide. 9-Nitrocamptothecin (9NC) is a potent topoisomerase-I inhibitor with strong anticancer effect. To increase the solubility and stability, we synthesized a novel 9NC loaded liposomes (9NC-LP) via incorporating 9NC into liposomes. In the present study, we determined the effects of 9NC and 9NC-LP on in vitro and in vivo, and the underlying mechanisms. Methodology/Principal Findings We first analyzed the characteristics of 9NC-LP. Then we compared the effects of 9NC and 9NC-LP on the proliferation and apoptosis of HepG2, Bel-7402, Hep3B and L02 cells in vitro. We also investigated their anticancer properties in nude mice bearing HCC xenograft in vivo. 9NC-LP has a uniform size (around 190 nm) and zeta potential (∼−11 mV), and exhibited a steady sustained-release pattern profile in vitro. Both 9NC and 9NC-LP could cause cell cycle arrest and apoptosis in a dose-dependent and p53-dependent manner. However, this effect was not ubiquitous in all cell lines. Exposure to 9NC-LP led to increased expression of p53, p21, p27, Bax, caspase-3, caspase-8, caspase-9 and apoptosis-inducing factor, mitochondrion-associated 1 and decreased expression of Bcl-2, cyclin E, cyclin A, Cdk2 and cyclin D1. Furthermore, 9NC-LP exhibited a more potent antiproliferative effect and less side effects in vivo. Western blot analysis of the xenograft tumors in nude mice showed similar changes in protein expression in vivo. Conclusions/Significance In conclusion, 9NC and 9NC-LP can inhibit HCC growth via cell cycle arrest and induction of apoptosis. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo, which means it is a promising reagent for cancer therapy via intravenous administration.

Aberrant expression of miRNAs is associated with particular cancers showing tissue- and clinical-feature-specificity patterns. Some miRNA genes harboring or being embedded in CpG islands undergo methylation mediated silencing. MBP, methyl CpG binding protein, suppresses transcription through binding to methylated CpG dinucleotides. Expression of miR-373 has been reported to be suppressed in malignant bile duct cell lines. Bioinformatic prediction reveals that the transcription start site (TSS) of miR-373 is implanted in a 402 bp canonical CpG island containing 26 CpG dinucleotides. In this study, we aim to determine the epigenetic regulation of miR-373 gene in hilar cholangiocarcinoma. Taqman microRNA assay shows that down-regulation of miR-373 is closely associated with poor cell differentiation, advanced clinical stage and shorter overall and disease-free survival in hilar cholangiocarcinomas. Methylation analysis shows that the promoter-associated CpG island is hypermethylated which is consistent with the inhibition of miR-373. Chromatin immunoprecipitation (ChIP) assay indicates that down-regulation of miR-373 results from the selective recruitment of MBD2 to methylated CpG islands. In contrast, MBD2 knock-down by use of a specific siRNA promoted the expression of miR-373. Reactivation of miR-373 by pharmacologic induction of 5-aza-CdR and trichostatin A (TSA) led to decreased enrichment of MBD2 at CpG island regions. Enhanced expression of exogenous MBD2 in stable QBC939 cells which stably express pGL4-m373-prom induces strengthened recruitment of MBD2. Our findings suggest that miR-373 is a methylation-mediated gene and the implication of MBD2 in methylation-mediated suppression of miR-373 plays an important role in tumourigenesis and development in hilar cholangiocarcinoma.

To evaluate the effect of protecting parathyroid glands in situ in the operation of total thyroidectomy by detecting parathyroid hormone after the operation.In the surgical team, 1019 consecutive patients with thyroid diseases were treated with total thyroidectomy. During the operation, parathyroid glands were protected in situ with correctly identifying the parathyroid glands, precisely dissecting its envelope and protecting its blood supply. Serum calcium level and parathyroid hormone were measured before and 24 hours after operation. The patients who had symptomatic hypocalcemia or hypoparathyroidism were given supportive treatment and followed-up.At least one of the parathyroid glands was preserved and remained in situ in all cases. Eighty-nine cases (8.7%) had decreased parathyroid hormone levels and 42 cases (4.1%) had complicated symptomatic hypocalcemia. The symptoms of hypocalcemia in all these cases could be controlled by supportive treatment, and serum calcium level and parathyroid hormone had all recovered 1 - 6 months later. If 3 and 4 parathyroid were conserved in situ, the postoperative complication rate was significantly lower than those with 1 and 2 parathyroid conserved (decreased PTH 69/999 vs 20/20, symptoms of hypocalcemia 25/999 vs 17/20, all P0.01).The techniques to protect parathyroid glands in situ are effective measure to prevent the postoperative hypoparathyroidism in total thyroidectomy.

To investigate the causes and the measures of prevention and cure of the dangerous complications (bleeding, pancreatic fistula, biliary fistula and death) after radical pancreatoduodenectomy (RPD) for periampullary malignant tumor.The rate and management of dangerous complications of 156 cases with RPD which were continuous performed by Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between January 2006 and June 2008 were analyzed retrospectively, including 97 males and 59 females with 37 - 79 years old, the mean age was 56.9 years old.Among the 156 cases with RPD, four patients had massive hemorrhage of gastrointestinal tract due to stress ulcer, two patients had bleeding in the pancreas-intestinal anastomosis after the operation, the rate of postoperative bleeding was 3.9% (6/156). One patient with massive hemorrhage of gastrointestinal tract due to stress ulcer had severe pulmonary infection and ARDS, and died of respiratory failure finally (the overall mortality rate was 0.7%) after ICU for two months. One patients with bleeding in the pancreas-intestinal anastomosis had pancreatic fistula (the rate of pancreatic fistula was 0.7%) 3 days after the second laparotomy to open the jejunum of the pancreas-intestinal anastomosis and make a transfixion of the bleeding points in the stump. Another patient who had the tumor located in the inferior segment of the bile common duct had biliary fistula 11 days after the operation (the rate of biliary fistula was 0.7%). Two patients with fistula had good recovery by expectant treatment of ultrasound-guided puncture and drainage.Prompt and effective treatment of the complications of bleeding, pancreatic fistula, biliary fistula could maximally decrease the perioperative death rate.

To explore and evaluate the combined conservative managements in the treatment of cervical chylous leakage.Thirty nine cases of cervical chylous leakage from June 1992 to June 2008 were retrospectively analyzed in this hospital. All of the 39 cases were cured by treating with conservative individualized therapy, including the applying of diet with high calorie, high protein and low fat and fatty food should only contains medium-chain triglycerides, total parenteral nutrition, keep the balance of hydrogen and electrolyte and correct hypoproteinemia, local pressure dressing, high persistent vacuum drainage (-50 approximately -80 kPa) and/or somatostatin analogue.All the cases of chylous leakage happened 2nd to 5th days after the operation. Among the 39 cases, 7 were high flow (drainageor=500 ml/d) chylous leakage, the amount of drainage reached as high as 1440 ml per day. The time of chylous leakage closure was 3 approximately 12 days, and the mean time was 7 days. No one experienced re-operation, wound hydrops or wound infection.The conservative individualized therapy may play a key role in the treatment of cervical chylous leakage.

AIM: To investigate the effects of exogenously mutated p27kip1 (p27) on proliferation and apoptosis of human cholangiocarcinoma cell line, QBC939 in vivo. METHODS: Adenoviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line. Expression of p27 was detected by RT-PCR. Western blot. Cell growth, morphological change, cell cycle, apoptosis and cloning formation were determined by MTT assay and flow cytometry. RESULTS: The expression of p27 protein and mRNA was increased significantly in QBC939 cell line transfected with Ad-p27mt. The transfer of Ad-p27mt could significantly inhibit the growth of QBC939 cells, decrease the cloning formation rate and induce apoptosis. p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Ad-p27mt. CONCLUSION: p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis. Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.

To investigate the technique of radical pancreaticoduodenectomy for malignant tumor in pancreatic head with pressed superior mesenteric blood vessel or portal vein.From March 2005 to March 2007, thin slice scan and vessel-reconstruction of 56 patients of malignant tumor in pancreatic head with pressed superior mesenteric blood vessels or portal vein were carried out using multidetector spiral CT to evaluate whether peripheral vessels of pancreatic tumor were invaded and whether the tumor was resectable. During the operation, 3 vascular blocking bands for superior mesenteric vein, portal vein and spleen vein or 4 vascular blocking bands (additional one for inferior mesenteric vein) were preset. Under the cross and traction between superior mesenteric vein and superior mesenteric artery, resected the uncinate process of pancreas thoroughly. Using those methods, radical pancreaticoduodenectomy for 56 patients above-mentioned were successfully accomplished.The accuracy for preoperative judging by using multidetector spiral CT whether the peripheral vessels of pancreatic cancer were invaded and whether the tumor was resectable was 98% and 100% separately. Thirty-seven of 56 patients, whose superior mesenteric blood vessels or portal veins were pressed by the tumor of pancreatic head, were operated using 3 vascular blocking bands and 2 patients using 4 vascular blocking bands, followed by suturing the bleeding points of the superior mesenteric vein with 5-0 vascular suture Proline. One patient's superior mesenteric vein was partially resected and restored. The operations cost 5-8 h each and the blood loss was 200-600 ml. There were no operative or postoperative hemorrhage or pancreatic juice leakage. According to the follow-up up to now, 2 patients died of multiple live tumor metastases 7 and 9 months separately after operation, the other 54 patients were still alive.Thin slice scan and vessel-reconstruction using multidetector spiral CT can accurately judge whether the blood vessels near the pancreatic tumor were invaded and whether the tumor was resectable, using 3 vascular blocking bands or 4 vascular blocking bands and cross, traction of the superior mesenteric blood vessels, operator can easily accomplish the radical pancreaticoduodenectomy of malignant tumor in pancreatic head with pressed superior mesenteric blood vessels and portal vein, which was not resectable or need combined resection of the blood vessels in the traditional opinion.

AIM: To explore the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the growth of biliary tract cancer cell lines (gallbladder carcinoma cell line and cholangiocarcinoma cell line) in vivo and in vitro, and to investigate the perspective of histone deacetylase inhibitor in its clinical application. METHODS: The survival rates of gallbladder carcinoma cell line (Mz-ChA-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazol tetrazolium (MTT) assay. A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, and changes in the growth of transplanted tumor after treated with TSA were measured. RESULTS: TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) in a dose-dependent manner. After the nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, the growth of cancer was inhibited in the model after treated with TSA. CONCLUSION: TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo.

Solid-pseudopapillary tumor of the pancreas (SPTP) is an uncommon and enigmatic pancreatic neoplasm that occurs mainly in young women. Although more and more cases have been reported in recent years, misdiagnosis and incorrect treatment still frequently take place. This study was designed to stimulate consideration of this tumor.We retrospectively reviewed the experience of diagnosis and treatment of 15 patients with SPTP and compared them with 516 patients with pancreatic cancer from January 1997 to March 2007.Most of the SPTP cases were asymptomatic except for one palpable mass. Almost all SPTPs demonstrated a solid structure with hypo- or iso-attenuation, cystic structure with hypo-attenuation on pre-contrast CT scan, and enhancement of solid portions on post-contrast CT scan. By contrast, most cases of pancreatic carcinoma had multiple symptoms and abnormal blood results. The tumors showed hypo-attenuation on both pre-contrast and post-contrast CT scan, and only a few showed iso-attenuation on post-contrast CT scan. All cases of SPTP in our group were cured by surgical resection, while only 16.86% of patients with pancreatic carcinoma could undergo a radical resection.Clinical features and CT scans were helpful to differentiate SPTP from pancreatic carcinoma. Radical surgical resection was the most effective and safe method for the treatment of SPTP.

The abnormal expression of atypical protein kinase C-iota (aPKC-iota) subtype and E-cadherin play important roles in tumor occurrence and progression. This study was designed to investigate the correlation of expression of aPKC-iota and E-cadherin with the clinicopathological characteristics and prognosis of cholangiocarcinoma, and to analyze the molecular mechanisms of invasion and metastasis of the tumor.EnVision immunohistochemistry was used to detect the expression of aPKC-iota and E-cadherin in 9 specimens of benign bile duct tissues, 35 specimens of cholangiocarcinoma and 6 specimens of metastatic cholangiocarcinoma. The relationship of the expression with clinicopathological characteristics, invasion and prognosis of cholangiocarcinoma was analyzed. A multivariate regression analysis was made of these data by the Cox proportional hazard model.The positive expression level of aPKC-iota in cholangiocarcinoma was remarkably higher than that in benign bile duct tissues (68.6% vs. 11.1%, P=0.006), but the expression level of E-cadherin was lower in cholangiocarcinoma than in benign bile duct tissues (37.1% vs. 88.9%, P=0.016). Correlation analysis revealed that the expression of aPKC-iota was positively related to tumor differentiation and invasion, whereas that of E-cadherin was entirely the contrary. Moreover, there was a negative relationship between the expression of aPKC-iota and that of E-cadherin (r=-0.287, P0.05). Univariate analysis showed that the overall survival rate of the group with a higher expression of aPKC-iota in cholangiocarcinoma was remarkably lower than that of the group with a lower expression (P0.01); multivariate analysis revealed that the expressions of aPKC-iota and E-cadherin are important prognostic factors for cholangiocarcinoma (P0.05).The expressions of aPKC-iota and E-cadherin may reflect the differentiation and invasive potential of cholangiocarcinoma. aPKC-iota and E-cadherin may be independent prognostic factors and, when used in combination with clinicopathological characteristics, may increase the accuracy in predicting the prognosis of patients with cholangiocarcinoma. As a polar regulative associated protein, aPKC-iota may play an important role in the invasion and metastasis of cholangiocarcinoma.

Proteomic analysis of bile fluid holds promise as a method to identify biomarkers of bile tract diseases, especially for tumors. Two-dimensional electrophoresis (2-DE) is a popular and proven separation technique for proteome analysis, but using this strategy for bile fluid analysis is still not fully developed. This study was undertaken to (a) establish a reliable method for general clean-up to make bile fluid samples suitable for 2-DE; (b) obtain 2-D biliary maps with high reproducibility and resolution; and (c) identify protein patterns present in 2-D biliary maps for potential tumor biomarker discovery, with the intention of distinguishing malignant from benign causes of bile duct obstruction.Bile fluid samples were obtained from two patients suffering from malignant and benign bile tract obstruction (one patient with cholangiocarcinoma as the experimental case, the other with cholelithiasis as control). A variety of sample preparation options, including delipidation, desalination and nucleic acid removal, were adopted to remove contaminants that affect 2-DE results. After that, each 350 microg purified sample was loaded onto nonlinear IPG strips (18 cm, pH 3-10 and pH 4-7) for first-dimension isoelectric focusing, and 12.5% SDS-PAGE electrophoresis for second dimension separation. Then 2-D maps were visualized after silver staining and analyzed with the Image Master 2-D software.A large number of protein spots were separated in 2-D maps from the experimental and control groups, with means of 250 and 216 spots on pH 3-10 IPG strips, and 182 and 176 spots on pH 4-7 strips, respectively. Approximately 16 and 23 spots were differentially expressed in matched pairs from the experimental and control cases using pH 3-10 and pH 4-7 strips.This study established a reliable sample preparation process suitable for 2-DE of bile fluid. By this method, 2-D biliary maps with high reproducibility and resolution were obtained. The differentially displayed proteomes in the 2-D biliary maps from the experimental and control groups indicated the potential application for bile fluid analysis to identify disease-associated biomarkers, especially for biliary tract tumors.

Studies showed that the abnormal expression of atypical protein kinase C iota subtype (aPKC-iota) and E-cadherin plays an important role in tumor genesis and progression. This study was to investigate the expression of aPKC-iota and E-cadherin in cholangiocarcinoma, and analyze molecular mechanisms of the invasion and metastasis of cholangiocarcinoma.The expression of aPKC-iota and E-cadherin in 9 specimens of benign bile duct tissues and 35 specimens of cholangiocarcinoma was detected by EnVision immunohistochemistry, and their correlations to the clinicopathologic characteristics and invasion of cholangiocarcinoma were analyzed.The positive rate of aPKC-iota was significantly higher in cholangiocarcinoma than in benign bile duct tissues (68.6% vs. 11.1%, P = 0.006), while the positive rate of E-cadherin was significantly lower in cholangiocarcinoma than in benign bile duct tissues (37.1% vs. 88.9%, P = 0.016). aPKC-iota expression was negatively correlated to E-cadherin expression (r = -0.287, P0.05). aPKC-iota expression was positively and E-cadherin expression was negatively correlated to the differentiation and invasion of cholangiocarcinoma (P0.05).The expression of aPKC-iota and E-cadherin may reflect the differentiation and invasive potential of cholangiocarcinoma. As a polar regulation-associated protein, aPKC-iota may play a role in the invasion and metastasis of cholangiocarcinoma.

Targeting is a new therapeutic tool for malignant tumor as a result of combining nanotechnology with chemotherapeutics. The aim of our study was to investigate the effects of magnetic nanoparticles enveloping a chemotherapeutic drug on human cholangiocarcinoma xenografts in nude mice.The human cholangiocarcinoma xenograft model was established in nude mice with the QBC939 cell line. The nude mice were randomly assigned to 7 groups. 0.9% saline or magnetic nanoparticles, including high (group 2), medium (group 4) and low (group 5) dosages, were given to nude mice through the tail vein 20 days after the QBC939 cell line was implanted. Calculations were made 35 days after treatment in order to compare the volumes, inhibition ratios and growth curves of the tumors in each group. Mice in each group were sacrificed randomly to collect tumor tissues and other organs for electron microscopy and pathological examination.The high and medium dosage groups were significantly different from the control group (P0.05). The tumor inhibition ratios for the high, medium and low dosage groups were 39.6%, 14.6% and 7.9%, respectively. The tumor growth curve of groups 5, 4, and 2 changed slowly in turn. The high and medium groups showed cell apoptosis under an electron microscope.Magnetic nanoparticles can inhibit the growth of human cholangiocarcinoma xenografts in nude mice.

To observe the pharmacokinetic and tissue-distribution characters of 5-flourouracil magnetic albumin deuto-microsphere (5-Fu-MAD) in normal and tumor-bearing mice, HPLC method for the determination of 5-Fu in plasma and tissues was established and applied to determine 5-Fu in mouse plasma and tissue samples. A Flame atomic absorption spectrometer was used to detect the iron concentration in mouse tissue. Plasma concentration-time curves of free 5-Fu, 5-Fu-MAD and 5-Fu-MAD plus the magnetic frame (MF) conformed to two compartment model of first order absorption and they had C(max) of 34.9, 7.95 and 5.97 mg x L(-1); T1/2 (Ke) of 22.26, 76.0 and 124.6 min, V(d) of 3.28, 30.7 and 66.1 L x kg; AUC(0-t), of 233.9, 78.3 and 50.2 mg x min x L(-1); AUC(0-infinity) of 237.2, 89.3 and 68.1 mg x min x L(-1), respectively. The distribution of 5-Fu and iron was the highest in the plenty blood perfusion organs like the liver, tumor, spleen and lung, while lower in the kidney and heart and lowest in brain and muscle. The tissue distribution of muscle and tumor increased significantly when a magnetic frame was inserted there. The pharmacokinetics and tissue distribution of 5-Fu-MAD exhibited sustained-release and target characteristics.

AIM To study the clinicopathological characteristics of unsuspected gallbladder carcinoma (UGC). METHODS We retrospectively studied 23 cases of UGC in Tongji Hospital, and compared their clinicopathological characteristics with 33 cases of preoperatively diagnosed gallbladder carcinoma (PDGC). RESULTS The proportion of UGC coexisting with cholecystolithiasis was significantly higher than that of PDGC (chi(2) = 13.53, P < 0.01). The infection rate of hepatitis B virus was 21.74% (5/23) in UGC and 30.30% (10/33) in PDGC. Nine (39.13%) of 23 patients with UGC and 8/33 (24.24) PDGC had contact with schistosome pestilent water. The rate of multiple pregnancies was 56.52% (13/23) in the patients with UGC and 42.42% (14/33) in PDGC. The primary location of the UGC was mostly in the neck and body of the gallbladder, and that of the PDGC was often in the body and bottom. The incidence of Nevin stage I and II UGC was significantly higher than that of PDGC (chi(2) = 4.44, P < 0.05 and chi(2) = 4.96, P < 0.05) while that of Nevin stage V UGC was significantly lower than that of PDGC (chi(2) = 7.59, P < 0.01). According to the grading of carcinoma, the incidence of well-differentiated UGC was significantly higher than that of PDGC (chi(2) = 4.16, P < 0.05), and that of poorly-differentiated UGC was significantly lower than that of PDGC (chi(2) = 4.48, P < 0.05). CONCLUSION There are different characteristics between UGC and PDGC, such as in primary location, malignant degree and incidence of coexistence with cholecystolithiasis. Cholecystolithiasis, hepatitis B, schistosome and multiple pregnancies were high risk factors for gallbladder carcinoma.

To investigate the effects of mutant exogenous P27(kip1) gene on chemosensitivity of human cholangiocarcinoma cell line.The recombinant vector was constructed and the mutant P27(kip1) gene was transfected into human cholangiocarcinoma cell line (QBC(939)). RT-PCR and Western blot were used to determine the expression of target genes. The effects of 5-fluorouracil (5-FU), mitomycin C (MMC) and cyclophosphamide (CTX) on the transfected cells were detected by assaying the apoptotic rate and growth inhibition by methabenzthiazuron (MTT) assay and flow cytometry (FCM).The mutant exogenous P27(kip1) gene was expressed effectively in the cells, and the expression enhanced the apoptosis and growth inhibition of QBC(939) inducted by 5-FU, MMC and CTX. The ratio of growth inhibiting increased significantly from 41.89% (5-FU), 45.59% (MMC), 38.91% (CTX) to 56.15% (5-FU), 55.65% (MMC), 51.69% (CTX), and apoptosis index from 13.76% +/- 3.03% (5-FU), 11.76% +/- 3.99% (MMC), 10.46% +/- 2.10% (CTX) to 41.39% +/- 4.32% (5-FU), 35.94% +/- 2.71% (MMC), 34.46% +/- 2.32% (CTX) (P0.05).The exogenous P27(kip1) gene transfer can remarkably increase the drug sensibility of the cholangiocarcinoma cells. The strategy targeted to control the cell cycle may be more effective in cancer treatment by combination of P27(kip1) gene therapy.

Hypermethylation of the promoter region is one of the major mechanisms of tumor suppressor gene inactivation. DNA methyltransferase 3b (DNMT3b), an enzyme that participates in the establishment of de novo methylation patterns, is highly expressed in many tumor cells and tissues, and it is closely associated with hypermethylation of the promoter of tumor suppressor genes. The aim of this study was to explore the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the expression of the DNMT3b gene in human biliary tract carcinoma cell.The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into the human biliary tract carcinoma cell line QBC-939 with lipofectamine transfection reagent, and positive cell clones were formed using G418 selection after transfection. The constructed recombinant plasmid was transfected into QBC-939 cells successfully and was confirmed by amplification of the exogenous neoR gene with the polymerase chain reaction method. The expression of DNMT3b gene mRNA and protein was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry respectively.Following transfection, the mRNA level of the DNMT3b gene decreased from 0.956+/-0.053 to 0.209+/-0.023, and the protein level of the DNMT3b gene also decreased from (75.38+/-3.22)% to (29.87+/-3.46)%. Very significant differences were observed both at the transcription and post-transcription levels in the expression of the DNMT3b gene between the non-transfection group and the antisense DNMT3b gene eukaryotic expression plasmid transfection group (P0.01).Transfection with the antisense DNMT3b gene eukaryotic expression plasmid can significantly reduce the expression level of the DNMT3b gene in the human biliary tract carcinoma cell line QBC-939. This study may provide a valid method to investigate the function of the DNMT3b gene and its role in biliary tract carcinoma.

The outcome of patients with cholangiocarcinoma is poor. To evaluate the experience in the diagnosis and surgical treatment of cholangiocarcinoma, we investigated the status quo of diagnosis and treatment of cholangiocarcinoma in China.The clinical data of 680 patients with cholangiocarcinoma treated at 8 hospitals from 1995 to 2001 were retrospectively analyzed with SPSS software package.The incidence of the tumor was the highest in the age group of 60-65 years. Meanwhile, the incidence was higher in aged men than in aged women, with a male to female ratio of 1.39:1. Proximal cholangiocarcinoma was the commonest (41.6%) and distant cholangiocarcinoma the second (28.7%) in the 680 patients. B-mode ultrasonography for cholangiocarcinoma was performed in 80.3% of the patients. Non-traumatic examinations such as computed tomography (CT), magnetic resonance image (MRI) and magnetic resonance cholangiopancreatography (MRCP) were more widely used than that of traumatic examinations such as percutaneous transhepatic cholangiography (PTC) and endoscopic retrograde cholangiopancreatography (ERCP). The low- and middle-differentiation cancer of the proximal bile duct accounted for about 50%. Most of the patients suffered from late-stage cholangiocarcinoma. The resection rate of the tumor was low, and the rate of radical operation was only 21.6% (147/680).Cholangiocarcinoma is common in the aged men. Its diagnosis and treatment have been improved, but little. Most patients are diagnosed as having late-stage cholangiocarcinoma at the time of outpatient clinic, and the rate of radical operation is low. Therefore, it is necessary to reinforce the early diagnosis and treatment of cholangiocarcinoma to improve the outcome after operation.

Fructose is cytoprotective during bile salt-induced apoptosis of hepatocytes by regulating protein kinase C (PKC). This study was undertaken to explore the regulating mechanism of hepatic injury in rats with obstructive jaundice, and to detect the PKC signal pathway.Rat hepatocytes were isolated by in situ collagenase perfusion and primary culture, and pretreated with various concentrations of PKC agonist phorbol myristate acetale (PMA) and inhibitor chelerythrine for 20 minutes. After pretreatment, 50 mumol/L glycochenodeoxycholate (GCDC) was added for additional 24 hours. Subsequently, the cells were detected by FCM and TUNEL. After adding with different concentrations of fructose and 100 mumol GCDC, the hepatocytes were evaluated by FCM and TUNEL. Experimental obstructive jaundice was induced with fructose and without fructose via double ligation of the bile duct for 3, 7, 14, and 21 days. Apoptotic status in the liver of all rats was detected with TUNEL, and PKC protein in the liver of obstructive jaundice (OJ) with the immunohistochemistry method.PMA increased GCDC-induced apoptosis and chelerythrine decreased GCDC-induced apoptosis in a concentration-dependent manner. Adding with different concentration of fructose and 100 mumol GCDC, the decreased apoptotic rate was related to the concentration of fructose. The apoptotic rate of the liver was related to times of OJ. PKC and apoptosis index (AI) were the highest after a 14-day ligation of the bile duct without use of fructose. AI and PKC were decreasing from a 14-day ligation of the bile duct with fructose.PKC takes part in the regulation, occurrence, and progression of hepatic injury in OJ. Fructose is cytoprotective during bile salt-induced apoptosis of hepatocytes by regulating PKC.

To study the role of Fas and Fas ligand (FasL) in biological behaviors of gallbladder carcinoma, and their correlated action and mechanism in tumor escape.Streptavidin-biotin-peroxidase immunohistochemistry technique was used to study the expression of Fas and FasL protein in 26 gallbladder carcinoma tissues, 18 gallbladder adenoma tissues, 3 gallbladder dysplasia tissues and 20 chronic cholecystitis tissues. Apoptosis of the infiltrating lymphocytes in these tissues was studied by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Expression of both proteins and apoptosis of the tumor infiltrating lymphocytes in cancer tissues of primary foci was compared with clinicopathological features of gallbladder carcinoma.The positive rates of Fas were not significantly different among carcinoma, adenoma, dysplasia and chronic cholecystitis. The positive rate of FasL in carcinoma was significantly higher than that in chronic cholecystitis (chi(2) = 4.89, P0.05). The apoptotic index (AI) in carcinoma was significantly higher than that in adenoma (t' = 4.19, P0.01) and chronic cholecystitis (t' = 8.06, P0.01). The AI was significantly lower in well-differentiated carcinoma and Nevin I-III carcinoma than that in poorly-differentiated carcinoma (t' = 2.63, P0.05) and Nevin IV-V carcinoma (t' = 3.33, P0.01). The confidence interval (CI) of infiltrating lymphocytes in adenoma, chronic cholecystitis, well-differentiated carcinoma and Nevin I-III carcinoma was very significantly lower than that in carcinoma (t' = 6.99, P0.01), adenoma (t' = 3.66, P0.01), poorly-differentiated carcinoma (t' = 5.31, P0.01) and Nevin IV-V carcinoma (t' = 3.76, P0.01), respectively. The CI of apoptosis of infiltrating lymphocytes in well-differentiated carcinoma was significantly lower than that in poorly-differentiated carcinoma (t = 2.52, P0.05), and was not significantly lower in Nevin I-III carcinoma than in Nevin IV-V carcinoma (t = 1.42, P0.05). Apoptosis of infiltrating lymphocytes was not discovered in adenoma and chronic cholecystitis.FasL expressed in gallbladder carcinoma cells permits tumor cells to escape from immune surveillance of organism by inducing apoptosis in infiltrating lymphocytes of carcinoma tissues. Up-regulation of FasL expression plays an important role in invasive depth, histological classification and metastasis of gallbladder carcinoma.

To study the effect of hepatitis C virus core protein (HCV-C) on human normal biliary epithelial cells (BEC) transformation and tumor development.BEC cells were transfected with plasmid pcDNA HCV-C (expressing HCV-C) by lipofectamine and selected in G418. The expression of HCV-C gene and protein was determined by PCR and immunohistochemical staining, respectively. Biological effect of transfected cells was observed through cell proliferation assay, anchor independent growth, and tumor development in nude mice. The expression of HCV-C protein in the induced tumor was evaluated by immunohistochemistry.HCV-C was strongly expressed in BEC cells transfected with plasmid pcDNA HCV-C and the positive signal was located in cytoplasm. The HCV-C expression protein in the induced cytoplasm. Cell proliferation assay showed that the population doubling time in the pcDNA HCV-C transfected cells was much shorter than that in the pcDNA3 and non-transfected cells (14 h, 28 h, 30 h respectively). The cloning efficiencies of transfected cells with pcDNA HCV-C, pcDNA3 and non-transfected cells were 36%, 2.5% and 1.5%, respectively (P0.01). Tumor developed in nude mice inoculated with pcDNA HCV-C transfected cells after the inoculation. HE staining showed bile duct carcinoma character and immunohistochemistry confirmed HCV-C expression in the tumor tissue. The positive control group also showed tumor development, while no tumor mass obtained in the nude mice inoculated with pcDNA3 and non-transfected cells even 36 days after the injection.HCV-C protein showed human normal biliary epithelial cells transformation and tumorigenic features.

To investigate the effects of dendritic cells (DCs) transfected with survivin gene, and to observe the effective and specific anti-tumor immunological effect induced by modified DC in vitro.Survivin gene was transfected to DCs with liposomes. Survivin expression could be detected both in DCs cells and in cell culture with method of Western blot. Cytokines as well as cellular surface molecule such as IL-12, TNF-alpha, CD1 alpha, CD83, MHCII, CD80 and CD86 were detected. The competence of inducing human specific cytotoxic T lymphocyte (CTLs) was also detected with MTT.Survivin expression could be detected both in DCs which were transfected with survivin cDNA and in cell culture superior. The IL-12 and TNF-alpha level was (265.2 +/- 32.7), (437.1 +/- 83.5) pg/ml, and much higher in transgened DC cells than blank DC cells (P0.05). CD1 alpha, CD83, MHCII, CD80 and CD86 was high expressed in survivin-DC cells, however, it was low expressed in blank DC cells. The lyse rate to gastric cancer cell, colon cancer cell and bile duct cancer cell was 65%, 77%, and 85% respectively, and these were much higher than those of blank DC cells.DCs transfected with survivin gene could induce specific cytotoxic T lymphocytes and strikingly raised DC cell's antigen present function, and have specific CTL killing activity.

Dendritic cells (DCs) are the most potent antigen-presenting cells and are actively used in cancer immunotherapy. Wild-type p53 can be recognized as an antigen and can induce specific cytotoxic T lymphocytes (CTLs) in the host body. The aim of this study was to investigate the effects of DCs transfected with full length wild-type p53 and modified by bile duct lysates on immune response.The wild-type p53 was transducted to DCs with adenovirus, which were modified by bile duct lysates (Lywtp53DC). The concentration of the surface molecules (B7-1, B7-2, MHC-I, MHC-II) of all DCs was detected with fluorescence activated cell sorter (FACS), and the ability of the DCs to induce efficient and specific immunological response in anti-(51)Cr-labeled target cells was studied. BALB/c mice infected with the DCs and QBC939 were used. CTL response in mice immunized with Lywtp53DC and treatment of tumor-bearing mice with Lywtp53DC and CTL response in these mice were studied.The surface molecules of Lywtp53DC had a high expression B7-1 (86.70%+/-0.07%), B7-2 (18.77%+/-0.08%), MHC-I(87.20%+/-0.05%), MHC-II(56.70%+/-0.07%) with FACS. The T lymphocytes had a specific CTL lysing ability induced by Lywtp53DC, with a CTL lysis rate of 81%. The immune protection of Lywtp53DC group was obvious, and the tumor diameter of the Lywtp53DC group was 3.10+/-0.31 mm, 2.73+/-0.23 mm, 3.70+/-0.07 mm on days 13, 16 and 19, smaller than those of any control groups (P0.05), DC, wtp53DC and LyDC. On the other hand, the growth rate of tumor of the Lywtp53DC group was slower than that of any other groups (P0.05).Dendritic cells transfected with wild-type p53 and modified by bile duct lysates have specific CTL killing capability.

Hepatitis C virus (HCV) is believed to be an important human pathogen causing carcinoma. But the effect of HCV infection on the alteration of cellular proliferation and apoptosis and the relationship between the effect and the development of hilar cholangiocarcinoma are largely unknown. The aim of this study was to assess the effect of HCV core protein on proliferation and apoptosis of hilar cholangiocarcinoma.HCV core protein (HCV C protein) was detected by peroxidase-antiperoxidase assay in surgical specimens from 48 patients with hilar cholangiocarcinoma. The apoptosis index (AI) and PCNA index (PI) in hilar cholangiocarcinoma were detected by in situ end labeling assay and streptavidin-biotin assay respectively.The expression of HCV C protein was observed in 32 (67.7%) of the 48 specimens of hilar cholangiocarcinoma. The mean+/-standard deviation for AI and PI was 3.52%+/-0.64% and 46.24%+/-11.46% respectively. The AI of hilar cholangiocarcinoma specimens with HCV C protein expression was significantly lower than that of HCV C protein negative specimens (P0.01), whereas the PI of HCV C protein positive specimens was significantly higher than that of HCV C protein negative specimens (P0.01).HCV C protein may promote the cellular proliferation of hilar cholangiocarcinoma and inhibit its cellular apoptosis.

To compare the efficacy of ceftriaxone and that of cefoperazone plus sulbactam (sulperazon) in controlling infection, in scavenging bacteria from bile, and in their costs when treating acute suppurative cholangitis with choledochostomy.Patients were randomly assigned to two groups: the ceftriaxone group (R-group, n=95) and sulperazon group (S-group, n=95). Before choledochostomy, both groups received one intravenous dose of the corresponding antibiotics: and 2 g ceftriaxnoe for the R-group, 2 g sulperazon, containing 1 g cefoperazone and 1 g sulbactam, for the S-group. After the operation, the patients in the R-group received ceftriaxone 2 g i.v. q.d.; the patients in the S-group received sulperazon 2 g i.v. b.i.d.. In addition, all patients in both groups received metronidazole 0.5 g daily before and after the operation. The efficacy was evaluated by efficiency in controlling infection and the persisting days of symptoms due to infection, fever and leukocytosis; the persisting days was compared using the life table method to calculate the "cumulative probability of persistence of symptoms (CPPS)". The two groups were also compared in regards to their biliary bacterial clearance rates and the costs directly attributable to the antibiotics.The efficiency in controlling infection was 98.9% (94/95) in both groups. However, the CPPS of the R-group decreased more rapidly than that of the S-group, Log-Rankchi2=6.7901, P=0.0092. Biliary bacterial clearance rate on post-operative day 3 was 72.0% (36/50) for the R-group, 41.3% (19/46) for the S-group, P=0.0037. Cost directly attributable to the antibiotics were (1788.29 +/- 518.46) yuan (RMB) for the R-group, and (3768.74 +/- 820.55) yuan for the S-group, F=395.51, P=0.0000.Both ceftriaxone and sulperazon are effective in treating acute suppurative cholangitis when used before and after choledochostomy. Ceftriaxone is superior in expediting symptom relief and bacterial clearance from bile, and is more cost-effective.

To study the effect of Hepatitis B virus X (HBx) gene transfection on expression of human telomerase reverse transcriptase (hTERT) mRNA in human bile duct carcinoma cell lines QBC939 and to elucidate the significance of cis-activation of hTERT mRNA by HBx gene on the carcinogenesis of bile duct.QBC939 were cultured in vitro and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using liposome-mediated gene transduction technique. Thirty six hours after transfection, EGFP expression, the indicator of successful transfection in cells, was determined. Flow cytometry was applied to determine the transfection efficiency. Cells were harvested and total RNA was extracted with TRI(ZOL) Reagent. The expression of hTERT mRNA in QBC939 was assayed by Reverse Transcription Polymerase Chain Reaction. The expression of HBx protein in QBC939 was detected by immunocytochemistry staining and western blotting.The transfection efficiency was 29.6% for both HBx expression vector and vector control group. The expression of hTERT mRNA was significantly increased when transfected with HBx expression vector than that transfected with OPTI-MEM medium and vector only. The expression of HBx protein could only be found in the cells when transfected with HBx expression vector by immunocytochemistry staining and western blotting.HBx gene transfection may up-regulate the transcriptional expression of hTERT mRNA in bile duct carcinoma cells. The cis-activation of hTERT gene by HBx gene is primary mechanism for carcinogenesis of biliary epithelia after HBV infection.

To observe the effect of octreotide on apoptosis rate of human pancreatic cancer cells PC-3 after transfected with somatostatin receptor type 2 (SST2) gene.SST2 plasmid was transfected into PC-3 cells by liposome. Result of transfection was detected by immunocytochemical staining and Western blotting. Apoptosis rates of PC-3 cells under different dosages of octreotide were measured by MTT assay and flow cytometry (FCM).Apoptosis rate caused by octreotide of transfected PC-3 cells was 7.56+/-1.06% at the dosage of 0.20 microg/mL, 9.25+/-1.73% at the dosage of 0.40 microg/mL and 14.18+/-2.71% at the dosage of 0.80 microg/mL. Apoptosis rate caused by octreotide of non-transfected PC-3 cells was 5.76+/-0.75% at the dosage of 0.20 microg/mL, 6.69+/-0.80% at the dosage of 0.40 microg/mL and 7.26+/-1.28% at the dosage of 0.80 microg/mL. Transfected PC-3 cells growth inhibition rate caused by octreotide was 9.36+/-1.34% at the dosage of 0.20 microg/mL, 12.03+/-1.44% at the dosage of 0.40 microg/mL and 20.23+/-4.21% at the dosage of 0.80 microg/mL. Non-transfected PC-3 cells growth inhibition rate caused by octreotide was 6.44+/-0.66% at the dosage of 0.20 microg/mL, 7.65+/-0.88% at the dosage of 0.40 microg/mL and 9.29+/-1.32% at the dosage of 0.80 microg/mL. We found that octreotide caused higher apoptosis rate and inhibition rate in transfected groups than in non-transfected groups (P0.05) at the tested dosages (0.20, 0.40 and 0.80 microg/mL).Deficiency of SST2 was probably the major reason why octreotide had little effect on PC-3 cells. Transfecting SST2 gene could strengthen the ability of octreotide of killing PC-3 cells. It provided an experimental evidence for using both octreotide and transfection with SST2 gene on clinical treatment of pancreatic cancer.

AIM: To study the transcriptional regulation of human telomerase reverse transcriptase (hTERT) mRNA in normal human cholangiocytes (HBECs) after hepatitis B virus X (HBx) gene transfection and to elucidate the possible mechanism of HBV infection underlying cholangiocarcinoma. METHODS: HBECs were cultured in vitro and co-transfected with a eukaryotic expression vector containing the HBx coding region and a cloning vector containing coding sequences of enhanced green fluorescent protein (EGFP) using lipid-mediated gene transfer. The transfection efficiency was determined by the expression of EGFP. The expressions of hTERT mRNA and HBx protein in HBECs were detected by RT-PCR and immunocytochemical stain, respectively. RESULTS: The transfection efficiencies were about 15% for both HBx gene expression plasmid and empty vector. No hTERT mRNA was expressed in HBECs when transfected with OPTI-MEM medium and empty vector, but a dramatic increase was observed for hTERT mRNA expression in HBECs when transfected with HBx expression vector. HBx protein was only expressed in HBECs when transfected with HBx expression vector. CONCLUSION: HBx transfection can activate the transcriptional expression of hTERT mRNA. Cis-activation of hTERT mRNA by HBx gene is the primary mechanism underlying the proliferation, differentiation and tumorigenesis of biliary epithelia.

To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing cholangiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis.QBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVec transfecting technique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective antibodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry.RT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proliferative index decreased significantly (P0.01), the percentage of S phase decreased remarkably (P0.05) in antisense vector transfected cells (9.27% +/- 1.91%) compared with that in QBC939 cells without transfection(16.35% +/- 2.87%), and the percentage of G0/G1 phase increased remarkably (P0.05) in antisense vector transfected cells (75.16% +/- 4.13%) compared with that in QBC939 cells without transfection (57.31% +/- 10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P0.05).Transfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholangiocarcinoma QBC939 cells.

To detect the expression of human telomerase reverse transcriptase (hTERT) protein and mRNA in bile duct carcinomas and the adjacent tissues and to elucidate its role in bile duct carcinogenesis.The expression of hTERT protein and hTERT mRNA in the formalin-fixed paraffin-embedded specimens of 71 cases of bile duct cancers and 39 cases of adjacent tissues was detected by streptavidin-peroxidase immunostaining and in situ hybridization. The correlation was analysed statistically between the expression of hTERT protein and mRNA and clinicopathological parameters bile duct carcinomas.The positive rate of hTERT protein expression and mRNA expression in malignant specimens was 78.9% (56/71) and 67.6% (48/71), while that in the adjacent tissues was 35.9% (14/39) and 23.1% (9/39), respectively. All the positive signals were found in the hyperplastic biliary epithelia. No significant correlation was established between hTERT expression and clinicopathological parameters.hTERT gene transcription and protein expression is most likely involved in the proliferation and malignant transformation of bile epithelia and the malignant progression of bile duct carcinomas. The detection of hTERT expression may serve elucidating the carcinogenesis of bile duct.

To detect the expression of HBV X gene (HBx mRNA) in extrahepatic biliary tract carcinomas and the adjacent non-cancerous tissues, and to analyzed the relationship between HBV infection and incidence of biliary tract carcinomas, thereby to elucidate the possible role of HBx in the carcinogenesis of biliary tract.The plasmid pSPX46 was digested by appropriate restriction enzyme. HBx fragment was obtained through gel extraction kit. The digoxigenin-labeled DNA probes for HBx mRNA were prepared by a random prime technique. The expression of HBx mRNA was detected in formalin-fixed- paraffin-embedded specimens from 71 cases of biliary tract carcinomas and 39 specimens of non-cancerous tissues adjacent to cancer by in situ hybridization. The correlations between HBx mRNA expression and clinicopathological parameters were statistically analysed in 71 cases of biliary duct carcinomas.Forty-three of 71 malignant specimens had detectable HBx mRNA expression with a positive rate being 61%. Only 7 of 39 specimens of non-cancerous tissues adjacent to cancer had weak HBx mRNA expression, with a positive rate being 18%, and all these positive signals were found in the hyperplastic biliary epithelium. No significant correlation was found between HBx mRNA expression and clinicopathological parameters, but a strong positive correlation was found between HBx mRNA and protein expression.There is a high frequency of HBx mRNA expression in extrahepatic biliary tract carcinomas. HBV infection and its gene integration might play a role to certain extent in the development of biliary tract carcinomas.

Since the resection rate is low for bile duct cancer and the drugs used for chemotherapy are less effective, we studied the inhibitory effects of 5-aza-2-deoxycytidine (ZdCyd) on bile duct cancer cell line QBC939 in vivo and in vitro and its possibility in clinical treatment.The survival and apoptosis rates of QBC939 after treatment with different dose of ZdCyd were detected by methyl thiazoy tetrazolium (MTT) and flow cytometry. The cooperative effect of ZdCyd with other chemotherapeutic drugs was also studied with MTT. The cancer cells were transplanted into nude mice, which were pre-treated with ZdCyd after tumor occurrence.ZdCyd decreased the cell survival rate, blocked the cell cycle at G1 phase, and increased the apoptosis rate. These effects were dose and time-dependent. ZdCyd also increased the anti-tumor effects of other chemotherapeutic drugs when used in combination. The tumor occurrence rate was lower in the ZdCyd pre-treated cells than in the untreated cells in nude mice, and ZdCyd was found to inhibit tumor growth.ZdCyd can inhibit the growth of QBC939 in vivo and in vitro through induction of cell apoptosis and has the cooperative effect on bile duct cancer cell when it is used with other chemotherapeutic drugs.

The inhibitory effect of antisense oligodeoxynucleotide (asODN) on the replication and expression of hepatitis C virus (HCV) in vitro is not well elucidated. This study was to assess the effect of asODN on HCV in cholangiocarcinoma.The QBC939 cells transfected by a recombinant HCV containing HCV core gene cloned in vector of PBK-CMV (PBK-HCVC) were treated by 14-mers phosphorothioate ODN complementary to the HCV core genomic region. The variation of HCVmRNA level was detected by RT-PCR. Moreover, the inhibitory effect of asODN was observed in nude mice.HCVmRNA was detected in transfected-QBC939 cells. The 14-mers complementary phosphorothioate ODN showed effective inhibition on HCVmRNA and unexpression HCVmRNA at 6 micromol/L. The tumorigenicity of the transfected-QBC939 cells incubated with asODN in nude mice was greatly inhibited.The results suggest a potential therapy of asODN for HCV infected cholangiocarcinoma.

To clarify the relationship between loss of DPC4 gene expression and pathogenesis of pancreatobiliary carcinoma.75 slides of normal duct (20), hyperplasia (15), dysplasia (15), invasive carcinoma (25) from patients with pancreatic diseases including pancreatic carcinoma (25 patients), chronic pancreatitis (6), pancreas injury (2) and 71 slides of common bile duct (CBD) carcinoma (38), gallbladder carcinoma (18), hilar bile duct (HBD) carcinoma (15) from patients with primary biliary tract carcinoma were analyzed for the expression of DPC4 protein by immunohistochemical staining.All specimens from 20 cases of normal duct and 15 cases of hyperplasia showed marked expression of DPC4 protein. The frequency of loss expression of the DPC4 gene was 33% in dysplasia, and 48% in invasive carcinoma. There was a significant statistical difference between hyperplasia and dysplasia (P0.01) and in dysplasia vs invasive carcinoma (P0.05). The frequency of loss expression of the DPC4 gene was 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, and 13% in HBD carcinoma. The frequency of loss expression of the DPC4 gene was significantly different in CBD carcinoma vs gallbladder carcinoma and HBD carcinoma (P0.01).Inactivation of the DPC4 gene occurs late in the neoplastic progression of pancreatic carcinoma. The frequency of DPC4 gene alternation was different in various locations of biliary tract carcinoma. In CBD carcinoma, this frequency is similar to that in pancreatic carcinoma, indicating their similar molecular alternations.

To investigate the expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in extra-hepatic cholangiocarcinoma and the relationship between their expression and clinicopathological parameters.COX-1 and COX-2 were detected in 56 extra-hepatic cholangiocarcinomas, including 31 matched tissues originating from non-tumorous bile ductal tissue adjacent to tumours and 6 normal bile ductal tissues, by immunohistochemistry strept avidin-biotin complex using isozyme selective antibodies.There was no difference in expression of COX-1 between carcinomas (96%, 54/56) and noncancerous specimens (94%, 29/31, P0.05) or normal bile ductal tissues (100%, 6/6, P0.05). The positive rate of COX-2 expression in extra-hepatic cholangiocarcinomas (86%, 48/56) was significantly higher than their matched tissues (39%, 12/31, P0.01) and normal bile ductal tissues (0%, 0/6, P0.01). Overexpression of COX-2 in extra-hepatic cholangiocarcinoma was related to the metastasis of lymph nodes, distant organs or tissues (P0.05) as well as the degree of tumour differentiation (P0.05).The overexpression of COX-2 plays a crucial role in the carcinogenesis and development of extra-hepatic cholangiocarcinoma, indicating that COX-2 may serve as a target for chemoprevention of extra-hepatic cholangiocarcinoma.

To study the correlation between human cholangiocarcinoma in the porta hepatis and the infection of hepatitis virus.Immunohistochemistry was used to detect HBxAg and HCV-C protein in formalin-fixed and paraffin-embedded samples taken from 68 patients with cholangiocarcinoma in the porta hepatis. The findings were reviewed against the clinical records of the patients.Six patients (8.8%) were positive for HBxAg and 24 (35%) for HCV-C protein, respectively. One patient was positive for both HBxAg and HCV-C protein. There were statistically differences in the extent of differentiation, invasion, lymph-node metastasis, and treatment between the patients with cholangiocarcinomas in the porta hepatis with HB(C)V infection and those without infection.HB(C)V infection is correlated to the development of cholangiocarcinoma in the porta hepatis. The tumor with HB(C)V infection may have a higher malignancy biologically and poorer prognosis. HBxAg and HCV-C protein may play an important role in the pathogenesis of cholangiocarcinoma in the porta hepatis.

To compare curative effects of various surgical procedures of bile duct stones.Two thousand nine hundred and fifty-five patients with intrahepatic bile duct lithiasis who had undergone various surgical procedures were analysed with Meta-analysis. Some of these cases were reported in Chinese Medical Journals from January 1990 to March 2001 and others were from Tongji Hospital.There was a significant difference between curative effects of non-hepatectomy and that of hepatectomy (chi(2) = 62.945, P0.01), and the outcomes of hepatectomy were much better than those of non-hepatectomy with OR(S) equalled to 0.303 (0.222 - 0.413). There was not a significant difference between curative effect of interposed jejunum and that of hepatectomy (95% CI of RR from 0.98 to 1.04). All the other operation, effects were worse than hepatectomy (upper limit of 95% CI of RR1).Hepatectomy is the most ideal surgery for intrahepatic bile duct stones and operation methods should be diversified since good effect could also be obtained when other operations are performed on suitable cases.

In order to study the diagnosis and treatment of HBV and HCV infection.We retrospectively analysed clinical data of 680 patients with cholangiocarcinoma from 1995 to 2001 and stated by SPSS software.(1) The fastigium of cholangiocarcinoma was 60 - 65 years old. The incidence of cholangiocarcinoma was higher in aged males and the sex ratio (male:female) was 1.36:1. (2) The proximal cholangiocarcinoma was most (41.6%) and distant cholangiocarcinoma was secondly (28.7%). (3) Most patients of cholangiocarcinoma were late. The resection rate was low and the rate of radical operation was 21.6% (147/680). (4) The incidence of proximal cholangiocarcinoma was higher in the positive Serologic marks for HBV and HCV and course of diseases was short. Moreover, the pathology of. positive Serologic marks for HBV and HCV trended to low-differentiation and invasion, metastasis and the resection rate was lower.Cholangiocarcinoma is common in the aged males. The infection of HB(C)V and hilar cholangiocarcinoma are correlated and incline to the proximal bile duct. The hilar cholangiocarcinoma infected HB(C)V may have higher malignant degree in biological characteristics and more badly prognosis.

AIM: To explore the effects of COX-2 gene in the proliferative activity induced by bile from anomalous pancreaticobiliary ductal union (APBDU) on human cholangiocarcinoma cell line. METHODS: Bile sample from APBDU and normal bile sample were used for this study. The proliferative effect of bile was measured by methabenzthiazuron (MTT) assay; COX-2 mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Cell cycle was analyzed by flow cytometry (FCM), and the PGE2 levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). RESULTS: Bile from APBDU can significantly promote the proliferation of human cholangiocarcinoma QBC939 cells compared with normal bile (P = 0.005) and up-regulated remarkably their COX-2 mRNA expression (P = 0.004). The proliferative activity of APBDU bile can be abolished by addition of cyclooxygenase-2 specific inhibitor celecoxib. CONCLUSION: Bile from APBDU can promote the proliferation of human cholangiocarcinoma QBC939 cells via COX-2 pathway.