ObjectiveTo investigate the effects of heterogeneous phosphatase and tensinhomologue deleted on chromosome ten (PTEN) on cell cycles,proliferation,invasion,tumorigenicity,metastasis and the expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR)proteins in human pancreas cancer cell line ( ASPC-1 ).MethodsASPC-1 cells was transfected with plasmid pE-PTEN containing PTEN,and empty plasmid pE-PTEN transfection was used as control,then ASPC-1-pE-PTEN (A-pE-P) cell and ASPC-1-pE (A-pE) cell was obtained.The expression of PTEN mRNA was determined by RT-PCR. PTEN,VEGF and EGFR proteins were measured by cell immunohistochemical method.Clone formation assay was used to observe the numbers of clone.Transwell was used to test the invasion ability of cells.The growths of tumor were detected by nude mice subcutaneous injection of cancer cells in vivo.Results Compared with ASPC-1,the expressions of PTEN mRNA of A-pE-P increased by 179.3％,and the expressions of PTEN protein were also significantly increased.The expressions of VEGF protein were significantly decreased.The expressions of EGFR protein were not significantly changed.Number of G2/M phase cells was significantly increased from (26.81 ± 1.03)％ to (31.5 ± 1.76)％ (P ＜0.05).The numbers of clone was decreased by 28％ (P ＜0.05).The number of penetrating cells was decreased[(46.3 ±6.6) vs (63.8 ±7.5) per high power field,P ＜0.05].The tumor volumes were significantly reduced [(142.4 ±30.9) vs (202.7 ±43.6) mm3,P ＜0.05].The tumor inhibitory rate was 42.4％.The distant metastases were significantly reduced [(2.0 ±0.7) vs (5.0 ± 1.3),P ＜0.01 ].Conclusions Heterogeneous PTEN can not only inhibit the proliferation,invasion and metantasis of ASPC-1 cells,arrest the cell growth at G2/M phase,but also decrease the expressions of VEGF.

ObjectiveTo investigate the methylation of the promoter region in miRNA in pancreatic cancer cell line PANC1 and normal pancreatic tissue,to discover the miRNA with hypermethylation associated with pancreatic cancer.MethodsThe genomic DNA of PANC1 and normal pancreatic tissue was extracted,and fractured by ultrasound.Methylation DNA fragments were obtained by 5-methyl of pyrimidine nucleoside antibodies and immunomagnetic beads.The hypermethylation miRNA differentially expressed between PANC1 and normal pancreatic tissue was selected by using methylation DNA chip.BSP ( bisulfite genomic sequencing PCR) and TA clone sequencing was performed for further validation.The genomic DNA of pancreatic cancer cell lines BXPC3,CFPAC1,PANC1 and SW1990 was extracted.The COBRA (combined bisulfite restriction analysis) was used to validate differentially expressed hypermethylation miRNA.ResultsEight differentially expressed hypermethylation miRNAs were screened from the DNA methylation chips,then five of them were selected for sequencing.The methylation status of miRNA-615,-663,-663b was significantly higher in the PANC1 than in normal tissues (60.6％ vs 7.6％,88.8％ vs 22.2％,94.4％ vs 13.0％ ) ; the methylation status of miRNA-675 was not significantly different between PANC1 and normal pancreatic tissue (76.0％ vs 100％ ).Due to large error in sequencing,miRNA1826 was excluded.The results of COBRA confirmed all the 4 miRNAs were highly methylated in PANC1 ; except for miRNA-675,other 3 miRNAs were highly methylated in BxPC,miRNA-663,miRNA-663b were highly methylated in CFPAC1,while miRNA-615,miRNA-663 were highly methylated in SW1990.ConclusionsHypermethylation miRNAs were differentially expressed between pancreatic cancer cell lines and normal pancreatic tissue,among them,highly methylated miRNA-663 was possibly associated with pancreatic cancer.

ObjectiveTo investigate the value of uncinate process first for pancreaticoduodenectomy (PD).MethodsThe clinical data of 19 patients admitted from December 2010 to March 2011,who underwent uncinate process first for PD were studied.ResultsAmong the 19 patients,there were 5 cases of periampullary adenocarcinoma,11 cases of pancreatic cancer,1 case of duodenum aggressive fibromatosis,1 case of main pancreatic duct type IPMN,1 case of SPN.During operation,3 patients (21％) were found to have abnormal or aberrant right hepatic artery.Among the 11 patients with pancreatic cancer,there are Peripancreatic lymph node(3 ～7) metastasis,in 7 cases,and nerve invasion occurred in 8 cases.All the N16 lymph nodes,pancreatic stump,bile duct margin,duodenum and retroperitoneal margin were negative,and all the cases were subjected to R0 resection.The median time for the portal vein blocking was 16 minutes.The average operation time was 4h and there was no major bleeding occurred,and the mean blood loss was 600 ml.No intractable diarrhea occurred post-operatively. Conclusions Uncinate process first for PD offers a comfortable,safe,accurate and controllable method to resect pancreatic head.

ObjectiveTo study the changes of serum levels of tumor n(ee)rosis factor-α (TNF-α),diamine oxidase (DAO),the expression of tight junction protein-1 (zonula occludens 1,ZO 1 ) in acute necrotizing pancreatitis (ANP) rats after ulinastatin intervention.Methods SD male rats were randomly divided into sham operation (SO) group,ANP group and ulinastatin treatment group.ANP model was induced by injecting 5％ sodium taurocholate into biliary and pancreatic duct.The rats were sacrificed at 6 and 24 hours,and then the levels of TNF-α,DAO and pathology change in pancreatic and intestinal were determined.The expression of bowel mucosa ZO-1 mRNA and protein was detected by RT-PCR and immunohistochemistry.ResultsSix hours after ANP induction,massive pancreatic necrosis and inflammatory cells infiltration were present,while epithelium necrosis of villi in ileum,vessel hemorrhage and inflammatory cells infiltration was found.The pathologic injury of pancreas and ileum in ulinastatin group was reduced when compared with that in ANP group.The serum levels of TNF-α were ( 10.83 ± 0.96),( 181.89 ± 4.93 ),( 128.23 ± 2.40) ng/L in SO group,ANP group and ulinastatin group; and the activities of DAO were (354.79 ±3.67),(117.21 ±5.58),(282.98 ± 9.12 ) U/L; the expressions of ZO 1 protein were 10.00 ± 1.87,1.20 ± 0.84,5.80 ±2.86; and the expressions ofZO 1 mRNA were 0.878 ±0.015,0.466 ±0.023,0.778 ±0.033.The serum level of TNF-α in ANP group was significantly higher than that in SO group,while the activities of DAO,expressions of ZO 1 mRNA and protein in ileum were significantly lower than that in SO group ( P ＜ 0.05 ).The serum level of TNF-α in ulinastatin group was significantly lower than that in ANP group,while the activities of DAO,expressions of ZO 1 mRNA and protein in ileum were significantly higher than that in ANP group (P ＜ 0.05).ConclusionsUlinastatin may inhibit the over release of TNF-α and improve plasma DAO activity,then increase the expression ofZO-1 mRNA and protein,thus protect the intestinal mucosa barrier.

Objective To detect the expression of aquaporin 1 in pancreas of rats with acute necrotizing pancreatitis (ANP) and to study the effect of Dachengqi decoction on it.MethodsOne hundred and sixty male SD rats were randomly divided into control group ( C group,n =32 ),ANP group ( n =32),Dexamethasone group (De group,n =32),Acetazolamide group (A group,n =32) and Dachengqi decoction group (DD group,n =32).ANP model was induced by retrograde injection of 5％ sodium taurocholate into the biliary and pancreatic duct.Rats in De group received dexamethasone (4 mg/kg) intravenously after ANP induction; while rats in A group received 1 ml acetazolamide via gastric lavage 2 h before ANP induction; rats in DD group received 2 ml Dachengqi decoction via gastric lavage 48,24,2h before ANP induction; rats in C group received laparotomy.Eight rats in each group were sacrificed at 3 h,6 h,12 h and 18h after induction of ANP models.Quantity of ascites and levels of serum amylases were measured.Pathological changes in pancreas tissue were detected by HE and electron microscope.Capillary permeability in pancreas tissue was detected by Evans Blue (EB) extravasations method.AQP1 expression in pancreas tissue was detected by real-time PCR and Western blotting.ResultsLevels of serum amylase in ANP group was significantly higher,and the pancreatic injuries were obvious ; the levels of serum amylase in De group and DD group was lower than that in ANP group,and the pancreatic injuries were attenuated.The levels of serum amylase in A group were higher than that in ANP group,and the pancreatic.injuries were more severe than that in ANP group.Six hours after ANP induction,the levels of EB in pancreas were (13.44 ±2.56),(126.35 ± 14.80),(86.31 ± 14.46),( 108.99 ± 15.07 ),(78.29 ± 16.85 ) mg/L In C group,ANP group,De group,A group and DD group,and the expression of AQP1 mRNA in pancreatic tissue was ( 170.07 ± 22.48 ) ％,( 83.93 ± 8.98 ) ％,( 117.09 ±10.70 ) ％,( 69.00 ± 8.98 ) ％,( 112.82 ± 11.79 ) ％ ; and the expression of AQP1 protein was 0.23 ± 0.06,0.10 ±0.02,0.32 ±0.03,0.13 ±0.02,0.45 ±0.04.The content of EB in ANP group was higher than that in C group,while the expression of AQP1 mRNA and protein in ANP group was significantly lower than that in C group (P ＜ 0.05 ).The content of EB in De group and DD group was significantly lower than that in ANP group,while the expression of AQP1 mRNA and protein was significantly higher than that in ANP group (P ＜ 0.05).ConclusionsAQP1 plays an important role in the pathogenesis of capillary endothelial barrier dysfunction in rats with ANP.Dachengqi Decoction can attenuate pancreatic injuries of rats by regulating the expression of AQP1.

ObjectiveTo establish nude mouse tumor models bearing human pancreatic adenocarcinoma SW1990 cells in multiple sites at different time-points and investigate the feasibiilty of multiple tumor-bearing in these models; then the findings and detection rate of 3.0T magnetic resonance image (MRI) in subcutaneous transplanted tumors was analyzed. Methods A total of 6 BALB/C nude mice were randomized into 3 groups (2 mice per group ).At the 1st,8th,15th day,the mice were injected subcutaneously with the suspension of SW1990 cells at left axilla and right axilla and right groin in sequence.Three weeks later,all the bearing-tumor mice were performed with MRI non-contrast enhanced scanning plus Gd-DTPA enhanced scan and the subcutaneous masses were subjected to pathological analysis.ResultsAll the 6 nude mice were alive during the study and obvious mass was observed in every injected site.The tumor size was positively associated with the grwing time.There were 9 tumors which could be de.ted by noncontrast enhanced MRI scanning and one more tumor was detected by contrast enhanced scanning.2 tumors were not detected,the 2 tumors were located at subcutaneous of right groin,with the shortest growing time,and the major axis of the 2 un-detected tumors was less than 5mm.Despite the MRI findings of the transplanted masses similar to that of human pancreatic adenocarcinoma with bleeding,necrosis,they presented the characteristics of a clear rim,with pseudocapeule sign.All the 12 masses were similar with human pancreatic adenocarcinoma under light microscope.ConclusionsIt is feasible to transplant human pancreatic adenocarcinoma cell at three different subcutaneous sites (injected at three different points of time) in the nude mouse,with a minimal survival time of three weeks.However,routine 3.0T MRI cannot detect the early tumors (growing time within 1 week,major axis ＜5 mm).

ObjectiveTo study the metabolite features of acute necrotizing pancreatitis (ANP) and chronic pancreatitis (CP) in rats.MethodsA total of 22 Wistar rats were divided into ANP group (n =7 ),CP group (n =6) and the control group (n =9).ANP model was induced peritoneous injection of 20％ Larginine,and the rats were sacrificed 12 hours later.CP model was induced by intravenously injection of DBTC (8 mg/kg body weight),and the rats were sacrificed after 2 months.The rats in the control group received same amount of saline.Serum amylase was determined and pancreatic tissues were pathologically examined.Metabolic changes of pancreatic tissues in vitro were studied by high resolution magic angle spinning nuclear magnetic resonance (MAS NMR ),and analyzed by using principal components analysis (PCA).Characteristic metabolites of ANP and CP were compared. Results Compared with the control group,increased leucine,iso-leucine and valine levels were observed in ANP group,however,the opposite trends were observed in CP group.Phosphocholine,glycerophosphocholine,choline levels were increased and fatty acids,lactate,betaine,glycine levels were decreased in both ANP and CP groups.The lipid content in CP group were significantly higher than that in ANP group and the increased taurine was only observed in CP group. Conclusions There were obvious metabolic features in pancreatic tissue in rats with pancreatitis disorders,and the increased taurine could be used as biomarker to discriminate ANP and CP.

Objective To evaluate the effects of infliximab (TNF-α monoclonal antibody ) on intestinal barrier injury in ANP complicated with MODS in a rat model.MethodsThirty SD rats were randomly divided into sham operation group (SO),ANP group and infliximab treatment group.Sodium taurocholate (4.5％) was injected into the pancreatic duct to induce ANP complicated with MODS model.Infliximab (8 mg/kg) was injected via tail vein in 6h after modeling in infliximab group.Same amount of 0.9％ NS was injected into the pancreatic duct in SO group.After 24 h of modeling,all rats were sacrificed,intestine and pancreas samples were collected for pathologic examination.The blood samples were harvested.The serum levels of amylase,TNF-α,diamine oxidase( DAO),D-lactate,and the rate of carbon propelling in ileum were measured.ResultsThe serum levels of amylase were ( 1125 ± 331 ),( 11024 ± 2203 ),( 545 ±30) U/L in SO group,ANP group and infliximab group; the serum levels of TNF-α were (12.1 ± 4.0),(107.6 ± 18.5),(75.8 ±5.9) U/L; the pathological scores of pancreas were 2.25 ±0.38,14.10 ±0.22,3.93 ± 0.67,the difference among the 3 groups was statistically significant ( P ＜ 0.05 ).The pathological scores of intestine were 2.29 ± 0.32,6.61 ± 0.58,3.91 ± 0.41 ; the DAO levels were ( 87.88 ± 34.51 ),(146.30 ±12.99),(115.00 ± 18.58) ng/ml; the D-lactate levels were (1.50 ±0.49),(2.32 ± 0.35),(2.02 ± 0.25 )mmol/L; and the rates of carbon propelling in ileum were (0.64 ± 0.04 )％,(0.28 ±0.08)％,(0.52 ±0.09)％,the difference among the 3 groups was statistically significant (P ＜0.05).ConclusionsInfliximab can effectively prevent dysfunction of intestinal barrier and improve motility in ANP rats.

Objective To evaluate 99mTc-ciprofloxacin (Infecton) scintigraphy as a method for detecting secondary infections associated with ANP in swine,in comparison with CT.MethodsTwenty-eight healthy swine were randomly assigned to control group (n =6),non-infected ANP (n =6) and infected ANP group( n =16).ANP model was induced by retrograde injection of sodium taurocholate and pancreatic protease mixture into the biliary and pancreatic duct.Two days after ANP induction,swine in infected ANP group were injected with 3 x 108 E.coli into pancreatic tissue,while swine in non-infected ANP group were injected with inactivated E.coli.At 7 d after inoculation,at 0.5,1,2,3,4,and 6 h after intravenous administration of 370 MBq of Infecton,SPECT scan was performed.Then 64-slice spiral CT scan was performed.Then swine were sacrificed,and histopathology examination and bacterial culture of pancreatic tissue were performed.The sensitivity,specificity,accuracy,positive predictive value and negative predictive value of the two methods to detect secondary infections were determined.ResultsThere were no abnormality in the normal pancreas and the bacterial culture was negative.There were pancreatic necrosis in the non-infected ANP group,but the bacterial culture was negative.There were pancreatic necrosis and infection in the infected ANP group and the bacterial culture was positive.The sensitivity,specificity,accuracy,positive predictive value and negative predictive value of the Infecton method were 93.8％ ( 15/16 ),91.7％ ( 11/12 ),92.9％ ( 26/28 ),93.8 ％(15/16) and 91.7％ ( 11/12),whereas these values for CT were 12.5％ (2/16),100.0％ ( 12/12),50.0％(14/28),100.0％ (2/2) and 46.2％ (12/26),respectively.The sensitivity,accuracy,and negative predictive value of the Infecton method were significantly higher than those in CT group (P ＜0.01 ).ConclusionsInfecton scintigraphy may be a better procedure for detecting ANP secondary infections than CT.

Objective To explore the expression and significance of hedgehog signal molecules (Ptch,Smo and Gli1 ) in chronic pancreatitis tissues in rats.MethodsSixty SD rats were randomly divided into CP group (n =50) and control group (n =10).DBTC solvent (8 mg · ml-1 · kg-1 ) was injected into the rat via tall vein in CP group.In control group,rats were treated only with the solvent at a dose of 1ml/kg body weight.All rats were sacrificed 6 weeks later to observe the pancreatic pathologic changes.Collagen accumulation in pancreatic sections was determined by staining for Sirius red.Expressions of Ptch,Smo,Gli1 mRNA and protein in pancreatic tissues were assessed by RT-PCR and immunohistochemistry.ResultsThe rate of chronic pancreatitis development in rats in CP group within six weeks was 73.9％.Collagen content was markedly higher in CP group than that in control group [ ( 38.52 ± 6.49 ) ％ ~s (7.37 ± 2.28 ) ％,P ＜ 0.05 ].No Path,Smo,Gli1 protein expression was observed in normal pancreatic tissues in control group.The positive rate of Ptch,Smo,Gli 1 expression was 73.5％,64.7％ and 52.9％ in CP group,and the difference between the two groups was statistically significant (P ＜ 0.05).The expressions of Ptch,Smo,Gli1 mRNA were 2.38 ±0.42,3.85 ± 1.03,4.63 ± 1.49 in CP group,which were significantly higher than those in control group (0.23 ±0.16,0.14 ±0.05,0.57 ±0.12,P ＜0.05).ConclusionsThe Ptch,Smo,Gli1 was highly expressed in pancreatic tissues in CP rats,suggests hedgehog messenger pathway may play an important role in the chronic inflammation and fibrosis of chronic pancreatitis.