Objective To detect the effect of osteopontin (OPN) siRNA on the growth,invasion and apoptosis of human gastric cancer cell line MKN28. Methods The eukaryotic expression vector of siRNA specific for OPN was constructed by liposome transfection,and the silencing effect was identified by fluorescence quantitative PCR and immunofluorescence staining. According to the inhibitory effect of the siRNA vectors,the experiment was divided into control group,non-specific OPN siRNA group,24-hour specific OPN siRNA group,36-hour specific OPN siRNA group,and 48-hour specific OPN siRNA group. The cell proliferation,apoptosis,migration and adhesion was finally detected among control group and OPN siRNA groups at different time points. Results For 48-hour specific OPN siRNA group,the expression of OPN in MKN28 cells was the lowest. Accordingly,the cell viability was decreased and the cell proliferation inhibitory rate was reduced to 30.20%; the ability of migration and adhesion was decreased,the number of cells attached on stromal cell membrane was much lesser (21.8?6.9 vs 42.0?9.4 in control group),and the ratio of cell adhesion on basement membrane matrix and fibronectin was lower [basement membrane matrix: (41.5?8.4)% vs (20.5?4.5)%; fibronectin: (25.3?4.5)% vs (14.6?2.5)% in control group],as well as the cell apoptotic rate was increased compared with the control group. Conclusion OPN gene silencing can inhibit the growth and invasion,as well as accelerate apoptosis in human gastric cancer MKN28 cells.

Objective To evaluate the diagnostic value of intraductal ulstrasonography(IDUS)in non-opaque bile duct stones.Methods Between January 2009 and August 2010 in the Department of Gastroenterology at Shanghai 10th People's Hospital,a total of 183 patients(male:102 cases,mean age 69 years； female:81 cases,mean age 71 years)were enrolled,who were suspected of bile duct stones or stenosis which could not be diagnosed by abdominal CT,MRI,and abdominal B-mode ultrasound.All the patients underwent endoscopic retrograde cholangiopancreatography(ERCP)first,and then patients with non-opaque bile duct stones followed by IDUS.Results A total of 134 cases (73.2％)of bile duct stones were diagnosed by ERCP,49 cases(26.8％)were negative.And then the 49 patients underwent IDUS,of whom 24 patients with sand-like stones,11 patients with lowdensity stones,6 patients with ampullary cancer,2 patients with pancreatic cancer,6 patients with sclerosing cholangitis.The diagnostic accuracy of IDUS in the position and quality of bile duct stones was 100％,higher than that of ERCP,which was 80％.After ERCP,pancreatitis occurred in 3 patients and improved after conservative treatment,there was no complications like perforation and bleeding.Conclusion The diagnostic accuracy of IDUS in the position and quality of bile duct stones is high,which can make up for the misdiagnosis by ERCP without increasing the complications.IDUS can provide reliable basis for the diagnosis of clinical bile duct stones.

Objective To study the expression of aquaporins-4 (AQP4) in the brain tissue of rats with pancreatic encephalopathy (PE) induced by phospholipase A2 and to explore the role of aquaporins-4 in PE.Methods Twenty five healthy Wistar rats were randomized into 3 groups:blank group ( n =5),PE group (n =10 ) and control group (n =10 ).The experimental model was established in rats by injecting phospholipase A2 into carotid artery (0.1 ml/100 g body weight).Same amount of normal saline was used in the control group and no treatment was used in the blank group.One day later,the rats were sacrificed,then the measurement of brain tissue wet/dry (W/D) weight ratio was performed,and brain tissue was routinely pathologically examined,immunohistochemistry and Western blotting were performed in each group to detect the expression of aquaporins-4.Results There was no obvious brain tissue pathological change in the control group and blank group.Neurons in the brain tissue of PE rats presented with significant edema and ballooning degeneration,infiltration of inflammatory cells,leukocyte aggregation around the microvessels.The water contents in the brain tissue in the blank group and control group,PE group were (61.44 ±0.36)％,(63.20±0.32)％ and (78.33 ±0.24)％,and it was significantly higher in PE group than that in the control and blank group (P＜0.05).The expressions of aquaporins-4 in the brain tissue were 0.41 ±0.27,0.49 ±0.13,0.98 ±0.21,respectively,and it was significantly increased in PE group than that in the control and blank group (P ＜ 0.05 ).Conclusions Aquaporins-4 may play important roles in the pathogenesis of pancreatic encephalopathy.

Objective To investigate the effects and mechanism of Safflower Injection on the proliferation,apoptosis,and expression of bcl-2/bax gene in hepatic stellate cell(HSC) in vitro.Methods HSC Line HSC-T6 was incubated with Safflower Injection at different concertration,cell proliferation was assessed by MTT colorimetric assay.After incubated with Safflower Injection 5,10,and 20 mg/mL,flow cytometry(FCM) was used to detect the content of DNA in HSC-T6.Morphological change of HSC-T6 was observed under microscope and agarose gel electrophoresis for DNA Ladder was used to detect apoptosis.Besides,the early stage of apoptosis was detected with Annexin V-FITC/PI double labbled assay.And real time RT-PCR was used to detect the expression of bcl-2/bax gene.Results The significant inhibition of Safflower Injection on HSC proliferation was observed in a dose-and time-dependent manner.Observed by FCM,the cell ratio in G0/G1 phase with Safflower Injection treatment(10 and 20 mg/mL) for 24 h was increased,which showed the significant difference compared with the control group(P

Objective To investigate the role of Hedgehog pathway in hepatic fibrosis and its association with activation of hepatic stellate cells. Methods Twenty male Spragur-Dawley rats were divided into control and model groups with 10 each. The animal models were induced by injection with CCl4 and fed with fat-rich diet. The rats in both groups were sacrified at the 8 week with 5 each and the liver tissues were removed for HSC-T6 culture. The deposition of collagen fiber in liver was detected with HE and Masson staining. RT-PCR was used to detect the expressions of Sonic hedgehog (Shh), smoothened (Smo), patched (Ptc), Gli-1 and α-smooth muscle action (α-SMA) mRNA in HSC-T6 and liver tissues. The influence of cyclopamine (Cyc) and lipopolysaccharide (LPS) on HSC-T6 proliferation were assayed by MTT. The expressions of Shh, Smo, Ptc, Gli-1 and α-SMA mRNA after intervention with Cyc (100μmol/L) and LPS were measured by real-time PCR. Results A lot of lipo and collagen deposited in liver of model rats. The Shh,Smo,Gli-1 and α-SMA mRNA were highly expressed in model rats than those in control group (2-△△Ct were 20.45±3.31 vs. 1, 12.78 ± 0. 53 vs. 1, 10.88 ± 2.41 vs. 1, 4.91 ± 2. 59 vs. 1, respectively, all P value <0. 05). In vitro Cyc inhibited HSC-T6 proliferation in dose dependant manner (F=636.81, P<0.01). Compared to the control group, the mRNA expressions of Smo, Ptc, Gli-1,α-SMA in HSC-T6 were significantly reduced after Cyc intervention (2△△Ct, were 0. 20±0. 11, 0. 21 ± 0. 08, 0. 28 ± 0. 05,0. 27±0.10,respectively, all P values<0.01). Conclusion The expression of members of Hedgehog pathway are increased in the progress of hepatic fibrosis, which may accelerate the hepatiee fibrosis by activating HSC.

Objective To investigate the role of pancreatic β cell on pancreatic regeneration following experimental acute pancreatitis.Methods Eighty-seven SD male rats were randomly divided into four groups:control group ( n =15 ),STZ group ( n =24),L-Arg group ( n =24 ),STZ + Arg group ( n =24).60 mg/kg of STZ was administrated by intraperitoneal injection to induce the diabetes model.2.5 g/kg body weight of LArg was administrated by intraperitoneal injection to induce the acute pancreatitis model.The rats were sacrificed 1,3,5,7 d later and the serum levels of amylase and glucose were measured.Relative pancreatic weight (pancreatic weight/body weight) were measured.Pancreatic tissue underwent routine pathologic examination,and the percentage of area of necrosis and tissue transformation was calculated.The expression of Reg4 and insulin was performed by immunofluorescence.Results Serum level of glucose significantly increased after STZ injection.After L-Arg injection,serum level of amylase significantly increased,and there was pancreatic tissue edema,necrosis,infiltration of inflammatory cells,which suggested the successful model induction.The percentage of area of necrosis in STZ + L-Arg group was (71.6 ± 6.0) ％ at the 3rd day,which were significantly higher than (42.3 ± 4.0 ) ％ in L-Arg group; the percentage of area of transformation was (45.6 ± 5.4) ％,which were significantly lower than (78.5 ± 6.4) ％ in L-Arg group.Expression of Reg4 in pancreatic islets of STZ + L-Arg group was significantly lower than those in L-Arg group.Conclusions STZ impairs pancreatic β cells,aggravates pancreatic damage following L-arginine induced pancreatitis and inhibits pancreatic regeneration.

Objective To investigate the expression of Iroquois homeobox protein 1 ( IRX1 ) gene and the hypermethylation status of its promoter in pancreatic cancer,and their relationship.Methods Real-time PCR was used to quantitatively detect IRX1 gene expression level of 12 sets of resected pancreatic cancer tissue and 6 sets of pancreatic cancer cell lines; gene sequences analysis was used to detect the structure of IRX1 promoter; DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) was used in pancreatic cancer cell lines,and then the methylation of IRX 1 was measured by methylation-specific PCR (MSP) and unmethylation sequence-PCR (USP) methods.Results Expression of IRX1 mRNA in pancreatic cancer tissue was 0.31 ± 0.11,which were significantly lower than that in normal pancreatic tissue ( 1.05 ±0.32,P ＜0.01 ).IRX1 mRNA expression of AsPCl,BxPC3,Capan 2,PANCl,PaTu8988 and SW1990 were 0.36 ± 0.08,0.34 ±0.16,0.37 ±0.11,0.25 ±0.06,0.31 ±0.04,0.36 ±0.02,which were significantly lower than that in human kidney epithelial 293 cells ( 1.03 ± 0.28,P ＜ 0.05 or ＜ 0.01 ).Analysis of IRX1 gene sequence showed abundant CpG islands in promoter.Hypermethylation of IRX1 promoter site was found in all pancreatic cancer cell lines.However,its methylation status could be reversed by 5-Aza-dC,and the IRX1 expression was also restored.Conclusions IRX1 mRNA expression is down-regulated in pancreatic cancer,and it is related with promoter CpG islands hypermethylation.

Objective To analyze expression of interleukin (IL)-23p19 and IL-23 receptor (IL-23R) in inflammatory bowel disease (IBD),and the role in the induction of peripheral blood T cell activation and proinflammatory cytokine secretion.Methods Peripheral blood (PB) and intestinal mueosal biopsies were collected from 12 patients with Crohn's disease (CD),25 patients with ulcerative colitis (UC) and 20 healthy controls.Expression of IL-23p19 was determined by immunohistochemistry and RT-PCR.IL-23R expression in CD4+,CD8+ T and NK cells from peripheral blood and lamina propria was analyzed by flow eytometry.Peripheral blood mononuclear ceils (PBMC) were isolated and cultured under stimulation with IL-23 and anti-CD3,and the levels of tumor necrosis factor α (TNFα) interferon (IFN)γ and IL-2 were determined by enzyme-linked immunosorbent assay (ELISA).Results The expression of II.-23p19 was significantly increased in inflamed mucosa of CD at both the transcriptional and translational levels compared with that in UC and healthy controls.IL-23R was mainly expressed in PB- and lamina propria-CD4+,CD8+T cells and NK cells from IBD patients,and markedly increased compared with controls (P＜0.05).IL-23 strongly triggered PBMC from IBD patients to produce significantly higher levels of IFN-γ,TNF-α and IL-2(P＜0.05).Conclusions IL-23p19 and IL-23R are highly expressed in IBD,particularly in CD,and may play an important role in the induction of T cell activation and proinflammatory cytokine secretion,suggesting that targeted therapy directed against IL-23 may have a therapeutic role in IBD.

Purpose To investigate relationship between PD-L1 molecule expression and Treg infiltration in non-small cell lung cancer ( NSCLC) tissue and to explore their clinical significance. Methods Immunohistochemistry was used to detect the PD-L1 molecules expression and Treg infiltration of 78 NSCLC tissues. The relationship among PD-L1 expression, Treg infiltration and clinic-pathological parameters was analyzed in the patients. Results PD-L1 molecule was highly expressed in lung cancer tissues than adjacent tissues (52. 5% vs 6. 4%), and same to Foxp3 +Treg (18. 63 ± 16. 67)/HPF vs (2. 96 ± 2. 97)/HPF. There was close relationship between PD-L1 expression and Treg infiltration with lymph node metastasis and clinical stage of patients, but was no statistical correlation with patient’ s age, gender, histological type and degree of differentiation of the tumor cells. PD-L1 expression was significantly higher in advanced stage than that in early stage (70. 0% vs 41. 7%) and same to Treg infiltration (73. 3% vs 35. 4%). There were also signif-icantly higher infiltration with lymph node metastasis than that without metastasis. In addition, PD-L1 molecule expression and Foxp3 +Treg infiltration were positively correlated (rs =0. 611, F=78. 82, P=0. 023). Conclusion There was strong relationship among PD-L1 expression, Treg infiltration and disease progress in lung cancer patients, and they possibly participate in the progression and immune escape of lung cancer.

Objective To study the correlation between non-alcoholic fatty liver disease (NAFLD)and glycosylated hemoglobin (HbA1 c) in middle-aged and aged population.Methods A total of 4 127 inservice workers and retirees aged 45 years old or above from one petrochemical enterprise in Ningbo were enrolled in our study.The waistline,body mass index,blood pressure,fasting blood-glucose,blood lipid profile,glutamyltranspeptidase,HbA1c and epigastrium B ultrasound were investigated.According to the quartile of HbA1c level,participants were divided into four groups,namely,Q1 group ≤5.2％,Q2 group ＞ 5.2％-5.4％,Q3 ＞ 5.4％-5.6％ and Q4 group ＞ 5.6％.The prevalence of NAFLD and clinical characteristics of each group were analyzed.Logistic regression was used to predict independent risk factors of NAFLD.Results The morbidity of NAFLD was 27.2％ with 31.9％ in male and 21％ in female,which was significantly higher in men.In Q1,Q2,Q3 and Q4 group,the prevalence of NAFLD were 18.5％ (178/961),22.8％ (185/812),25.6％ (280/1 095),38.1％ (480/1 259) respectively.With the increase of HbA1 c level,the morbidity of NAFLD increased synchronously.The age,systolic pressure,total cholesterol,low densitylipoprotein cholesterin and fasting blood-glucose were all elevated according to the increase of HbA1 c in 1 123 NAFLD patients.Multi-factor logistic regression analysis indicated that high HbAlc level was the risk factor of NAFLD (OR =1.67,95％ CI 1.15-2.43,P =0.007).Conclusion HbA1c is an independent risk factor of NAFLD and both of these are closely related to blood lipid metabolism disorder.