Objectives To verify if chemotherapy drug gemcitabine could induce a change of protein expression in pancreatic cancer cell line SW-1990, and to provide evidence for clinical therapy. Methods We use two-dimensional gel eleclrophoresis(2-DE), mass speclrornetry and bio-information methods to compare the change of protein expression before and after gemcitabine and 5-FU treatment of cell line SW-1990, and identify the discrepant protein expression. Results The concentration of gemcitabine, that can suppress 50 percent of the pancreatic cancer cells was 1-100 ng/ml, while same suppression for 5-FU was 250-2500 ng/ml. Six discrepant protein expressions, one in 5-FU group and 5 in gemcitabine group were found. Conclusions Gemcitabine has better therapeutic effects on the growth of pancreatic cancer cell line SW-1990 than that of 5-FU. Gemcilabine was involved in sugar and fat metabolism, and it could induce some changes of protein expression, for example ?-actin and MGC: 19713, which is of somewhat importance for clinical investigation of pancreatic cancer therapy.

Objective To investigate the dynamic changes in expression of cell markers desmin, glial fibrillary acidic protein (GFAP), and (-smooth muscle actin (?-SMA) in primary cultures of human and rat pancreatic stellate cells (PSCs). Methods PSCs were isolated from human as well as rat pancreas using Nycodenz discontinuous density gradient centrifugation following digestion with combination of collagenase IV, Pronase E and DNase I, and purified by centrifugal elution techniques. Freshly isolated cells were examined by laser scanning confocal microscopy for vitamin A autofluorescence, by immunostaining for desmin, GFAP, and ?-SMA. Expression of ?-SMA was as well measured by Western analysis. Procollagen ?1(Ⅰ) mRNA expression was analyzed by Northern analysis. Results The purity of rat PSCs obtained by centrifugal elution were above 95%. More than 85% of either freshly-isolated human or rat PSCs displayed positive vitamin A autofluorescence. Rat PSCs stained positively for desmin and GFAP and negatively for ?-SMA, whereas human PSCs were negative for either desmin, GFAP or ?-SMA. During the process of primary culture, rat PSCs were positive for ?-SMA at 3d and completely transformed from quiescent state to myofibroblast-like phenotypes at 7d, which negatively or scarcely expressed desmin and GFAP, but fully expressed the ?-SMA protein and procollagen ?1(Ⅰ) mRNA, similarly to the settings of human PSCs. Conclusions Human and rat PSCs could be successively isolated in above 95% purity by combining gradient centrifugation with following centrifugal elution techniques. The results show some species differences in desmin and GFAP expression between freshly-isolated human and rat PSCs. Both of which, however, acquire a myofibroblast-like phenotype largely expressing ?-SMA protein and procollagen ?1(Ⅰ) gene in culture.

Objective To determine the cleaning and the shortest disinfectant time for gastroscope Methods 1? g/ml HBsAg and 3? 108/ml bacteria of Aureus Staphylococci(ATCC 6538),Escherichia coli(ATTC 8099),Bacterium earuginosum(ATCC 27853)were artificially spreaded on the body of gastroscope ,biopsy hole and biopsy clamp.The gastroscope was routinely cleaned and disinfected.HBsAg and pathogens in the samples,which collected before and after cleaning the gastroscope and 3min,5min,10min after disinfection,were tested by using ELISA and bacteria culture,respectively.Results Pathogen and HBsAg can be detected before cleaning the gastroscope.Pathogen can also be detected,but HBsAg disappears after cleaning.Both HBsAg and pathogen was negative 3 min after gastroscope was immersed in glutaraldehyde.Conclusion The tests for pathogens and HBsAg can be negative after gastroscope was cleaned and immersed in 2％ glutaraldehyde for 3 min.

We prepared bovine epidermal keratin (EK) antibody, which can be used as a specific marker of cholangiocarcinoma(CC) cells to be distinguished from hepatocellular carcinoma (HCC) cells. The intrahepatically implanted solid tumors of FSK 7901 and FSK 7902 have the histological structures of CC and HCC, respectively. Using immunofluorescence and PAP techniques, we demonstrated that, in the solid tumors, FSK 7901 cells are definitely EK-positive, while most of the cells of FSK 7902 are EK-negative. Therefore, we considered FSK 7901 and FSK 7902 as CC and HCC, respectively. In the solid tumors of FSK 7902, few cells which are EK-positive may be those in which there are Mallory bodies or the structures are forming.

Objective To investigate the expression of peroxisome proliferator-activated receptor-γ(PPAR-γ) and angiotensin receptor (AT2R) in chronic pancreatitis tissue. Methods Between April 2006 and February 2009, 24 patients were pathologically diagnosed as chronic pancreatitis were enrolled and 12 samples of normal pancreatic tissues were treated as controls. Immunohistochemical staining was used to detect PPAR-γ and AT1R, AT2R expression in pancreatic tissues. Results PPAR-γ and AT1R were mostly negatively expressed in normal pancreatic tissues, the positive scores were 0.33±0.49 and 0.42 ± 0. 51, respectively, while AT2R were weakly positively or negatively expressed in acinus and islet cell, and it was weakly positively expressed in ductal epithelial cells, the positive score was 2. 33 ± 1. 37. In chronic pancreatitis tissue, PPAR-γ, AT1R and AT2R were positively expressed in acinus, ductal epithelial cells, and islet cell, the positive scores were 3.28 ± 2.46, 4.36 ± 2.80 and 4.61 ± 2.89, which were significantly higher than those in the normal group (P<0.01, P <0.01, P<0.05). Conclusions PPAR-γ, AT1R and AT2R were positively expressed in chronic pancreatitis tissue, and they may be the treatment target of chronic pancreatitis.

Objective To investigate Toll-like receptor-4 (TLR4) protein expression in human pancreatic adenocarcinoma,and to evaluate the relationship between TLR4 protein expression and angiogenesis.Methods Sixty-two surgically resected human pancreatic adenocarcinoma specimens and 35 normal para-cancerous tissues were investigated for TLR4 protein expression by immunohistochemical SP methods,and CD31 antibody was used to mark microvascular endothelial cells and determine the microvessel density (MVD).The correlation among TLR4 protein expression and MVD and clinicopathologic features of pancreatic adenocarcinoma were analyzed.Results TLR4 protein positive expression rate and MVD in human pancreatic adenocarcinoma was 74.2％ (46/62) and 47.3 ± 13.5,respectively,which were significantly higher than those in the normal pancreatic tissue [17.1％ (6/35),12.6 ±4.8; P ＜0.01].TLR4 protein positive expression rate in the cases with lymph node metastasis was 83.8％,which was significantly higher than that in the cases without lymph node metastasis (60.0％,P =0.036).TLR4 protein positive expression rate in the patients with stage Ⅲ and Ⅳ of TNM classification was 85.3％,which was significantly higher than that in the patients with stage Ⅰ and Ⅱ (60.7％,P=0.028).MVD was closely related to tumor size,lymph node metastasis and TNM stage of pancreatic adenocarcinoma (P =0.008,0.036,0.010).There was a strong positive correlation between TLR4 protein expression and MVD (r =0.534,P ＜0.01 ).Conclusions TLR4 protein expression is closely related to the development and progression of human pancreatic adenocarcinoma and its potential mechanism is related to the promotion of tumor angiogenesis.

Objective To investigate the diagnostic value of determination of the genotypes in codon 12 and 13 of K-ras oncogene in blood samples of patients with pancreatic carcinoma(PC).Methods Blood samples were obtained from 54 patients with pathologically confirmed PC,and 33 healthy controls.The DNAs were obtained in these samples.and then genotype of K-ras mutation was detected by using the PNA-clamping real-time quantitative PCR.Then the correlation between the K-ras genotypes of blood DNA and the clinical characteristics was analyzed.Results K-ras mutations were found in 74.1%(40/54)of patients with PC.There was no such mutation in control samples.The mutations of K-ras was associated with age,lymph node and vessel invasion.poorly differentiated tumor,CA19-9,while it was not associated with sex,tumor location,size of tumor,clinical staging and pathological type.Conclusions The one-step method was highly sensitive for detecting K-ras mutation in blood samples.Detection of circular blood cells harboring K-ras mutation suggested the tumor was highly invasive with poor prognosis.

Objective To explore the potential role of monocyte chemotactic protein-1 ( MCP-1 ) gene in the pathogenesis of acute lung injury (ALI) in early acute necrotizing pancreatitis (ANP). Methods Forty SD rats were randomly divided into control group, ANP 3, 6, 12 h group with 10 rats in each group according to a number table. ANP was induced by intraperitoneal injection of 15% L-arginine solution at a dose of 2.0 mg/g body weight. Pathological changes of pancreases and lungs were observed. Lung wet/dry weight ratio was measured. Intrapulmonary expression of MCP-1 mRNA was evaluated by RT-PCR. Results After intraperitoneal injection of 15% L-arginine solution, the rat's pancreas presented with bleeding, necrosis comparable with pathological changes of ANP. Pulmonary tissue edema was obvious. At ANP 3, 6, 12 h group, the pathological scores of the lung were 3.75±0.58,5.50 ±0.63,5.86 ±0.54, the wet/dry weight ratios were 4.85 ±0.38,4.97 ± 0.47,5.03 ± 0. 46, the MCP-1 mRNA expressions were 0.36 ± 0.08, 0. 56 ± 0. 15, 0. 72 ± 0.21,which were significantly higher than those in the control group (0.12 ±0.05,4.32 ±0.33,0.21 ±0.05, P＜0.05 or ＜0.01 ). The MCP-1 mRNA expression in lungs was significantly correlated with the degree of lung damage and wet/dry weight ratio of lungs (r=0.75,r=0.89,P＜0.05).Conclusions MCP-1 mRNA expression was up-regulated in the early phase of ANP in the lungs, and it may play an important role in ALI during ANP.

ObjectiveTo investigate the protective effects of p38MAPK inhibitor SB203580 on acute necrotizing pancreatitis associated lung injuries in rats.Methods Fifty-four SD male rates were randomly divided into 3 groups,including control group,ANP group,SB203580 group with 18 rats in each group.ANP was induced by intraperitoneal injection of L-arginine solution. Rats in control group were intraperitoneally injected with same amount of saline.Before ANP induction,the rats in SB203580 group received 10 μmol/L SB203580 dissolved by dimethyl sulfoxide at a dose of 5mg/kg weight via intraperitoneal injection.The rats were sacrificed at 3,6,and 12 h after operation,the serum levels of amylase,TNF-α,IL-6 was determined.Pathological changes of pancreas and lung were observed.The wet/dry (W/D) weight ratio of lung and MPO were measured.CINC mRNA of lungs was determined by RT PCR. Expression of phosphated-p38MAPK (p-p38MAPK) protein was evaluated by Western blotting.ResultsThe serum levels of amylase,TNF-α,IL-6and wet/dry (W/D) weight ratio of lung,MPO activity,CINC mRNA and p-p38MAPK protein expression of lungs were (1035 ±73)U/L,(0.94 ±0.16)μg/L,(4.77 ±0.86) μg/L,3.92 ±0.29,(0.39 ±0.02)U/g,0.28 ±0.04,0.09 ±0.04 in control group at 6 h after operation,and the corresponding values were (5848 ±656) U/L,(3.84 ±0.32)μg/L,(103.54 ± 15.32)μg/L,4.97 ±0.47,(1.03 ±0.08) U/g,0.62 ±0.06,0.52 ±0.14 in ANP group,while they were (4259 ±286) U/L,( 1.64 ±0.21 ) μg/L,(76.56 ± 11.46) μg/L,4.32 ±0.34,(0.78 ±0.05)U/g,0.37 ±0.04,0.27 ±0.08 in SB203580 group.The values in ANP group were significantly higher than those in control group,and the values in SB203580 group were significantly lower than those in ANP group,but they were still significantly higher than those in control group ( P ＜ 0.01 ).ConclusionsSB203580 may attenuate injury of lung and pancreas in ANP by blocking p38MAPK signal transduction pathway,and decreasing the production of inflammatory cytokines.

Objective To investigate whether determination of CEA and CA19-9 levels in EUS-FNA pancreatic samples can be useful in detecting pancreatic adenocarcinoma and differentiating pancreatic adenocarcinoma from chronic pancreatitis.Methods Levels of CEA,CA19-9 were examined by chemiluminescence immunoassay analysis in EUS-FNA specimens obtained from 25 patients with chronic pancreatitis and 65 patients with pancreatic adenocarcinoma,and compared with those of their peripheral serum.Twelve patients with suspected pancreatic adenocarcinoma while with negative EUS-FNA pathological findings were followed up.Results First,the levels of CEA,CA19-9 in EUS-FNA specimens were higher than those in serum obtained from same patient with pancreatic adenoearcinoma(P＜0.01),but there was no difference in these variables of EUS-FNA specimens and serum obtained from patients with chronic pancreatitis.Second,in the EUS-FNA samples,the levels of CEA,CA19-9 in pancreatic adenocarcinoma were higher than those in chronic pancreatitis(P＜0.01).On the contrary,in serum samples,there was no significant difference in CEA level between pancreatic adenocarcinoma and chronic pancreatitis(P=0.079).CA19-9 level in serum of Dancreatic adenocarcinoma was higher than that of chronic pancreatitis(P＜0.01). Finally,during the follow-up,of all the 12 patients with suspected pancreatic adenocarcinoma,10 patients were diagnosed as having pancreatic adenocarcinoma and 2 patients as having chronic pancreatitis.Diagnostic accuracy of serum CEA and CA19-9were 30%and 70%respectively,while sensitivity of CEA and CA10-9 determined by EUS-FNA was both above 90%.Conclusion The method of measuring CEA and CA19-9 levels in samples obtained by EUS-FNAcan be useful in detection of pancreatic adenocarcinoma and differentiation of malignant pancreatic tissue from chronic pancreatitis.