Expression and promoter methylation status of leucine rich repeat containing 55 in pancreatic carcinoma tissue and the clinical value / 中华胰腺病杂志

Objective:To detect the mRNA expression and methylation status of leucine rich repeat containing 55(LRRC55) gene in pancreatic carcinoma tissues, and discuss the clinical value.Methods:Resected pancreatic ductal adenocarcinoma and normal adjacent specimens from 37 patients admitted in General Surgery of First Affiliated Hospital of Naval Medical University were collected from May 2019 to May 2021. Another two normal pancreas specimens and two blood samples from healthy adults were also collected. All patients′ age, gender, tumor location, tumor size, tumor differentiation, TNM staging, lymphatic metastasis, CEA and CA19-9 level were recorded. Bisulfite treatment of genomic DNA and sequencing analysis was used to study methylation patterns in CpG islands of the promoter for LRRC55 gene in fresh tissues from 2 pancreatic adenocarcinoma and adjacent tissues, 2 normal pancreatic tissues, 2 pancreatic cancer cell lines (PaTu8988 and ASPC1). LRRC55 mRNA in 35 pancreatic adenocarcinoma and adjacent tissues was detected by real-time quantitative PCR and the correlations with clinical parameters were analyzed.Results:CpG islands of LRRC55 in pancreatic adenocarcinoma tissues and pancreatic cancer cell lines was highly methylated and the mean methylation rate was 53% and 71%, respectively; while LRRC55 gene in pancreatic adjacent tissues and normal pancreatic tissues was lowly methylated, and the mean methylation rate was 8% and 11%. The relative expression in the pancreatic adenocarcinoma tissues and the paired adjacent normal tissues was 0.21 (0.02, 1.00 ) and 0.98 (0.33, 3.66 ), respectively; the former was significantly lower than the later and the difference was statistically significant ( P=0.003). Correlation analysis showed that LRRC55 mRNA expression level was related to tumor differentiation and CEA, but not correlated with patients′ age, gender, tumor location and size, CA19-9 level, lymphatic metastasis and TNM staging. Conclusions:Pancreatic cancer tissue and cell lines had abnormal methylation of LRRC55 gene; LRRC55 gene hypermethylation was related with its lower mRNA expression level in pancreatic cancer, which was correlated with the tumor differentiation and CEA level. LRRC55 may be a potential suppressor gene for pancreatic cancer.

The expression of HMGB in rats with acute necrotizing pancreatitis / 中华胰腺病杂志

Objectives To detect the expression of serum high mobility group box-1 (HMGB1) and explore its changes in rats with acute necrotizing pancreatitis (ANP).Methods Intraperitoneal injection of 20％ L-arginine in the dosage of 250 mg/100 g twice every 1 hour was used to establish ANP rat model.Intraperitoneal injection of normal saline solution in equal volume was performed in control rats.Rats were sacrificed at 6 h,18 h,24 h,36 h,48 h,72 h and 96 h after injection.Blood samples were collected to detect serum amylase and HMGB1 level.Pancreatic tissue was collected for pathological examination.Realtime PCR was applied to detect the mRNA expression of HMGB1 in pancreatic tissue.Werstem blot was used to determine HMGB1 protein expression in pancreatic tissue.Results Serum amylase level began to increase at 6 h after modeling,reached the peak at 18 h [(5 070 ± 603) U/L] and returned to normal level after 48 h.Serum amylase activity at 6 h and 18 h in ANP group was much higher than that in control group (1 844 ± 181)U/L(P＜0.05).The expression of HMGB1 began to increase at 6 h,reached to the peak at 36 h [(288.5 ±42.1)μg/L],and then decreased gradually.HMGB1 expressions at each time point in ANP group were significantly higher than those in control group (31.6 ± 10.1) μg/L],and the differences were statistically significant (all P ＜ 0.05).Pathological scores in pancreatic tissues in ANP group were higher than those in control group 0.38 ± 0.52,and the differences were statistically significant (P ＜ 0.05).HMGB1 mRNA expressions at t 6 h,18 h,24 h,36 h,48 h,72 h and 96 h in ANP group were 1.23 ±0.25,2.60 ± 0.46,3.23 ± 0.34,4.77 ± 0.66,2.88 ± 0.56,2.05 ± 0.20,1.33 ± 0.28,which were significantly higher than those in control group 0.44 ± 0.09,and the relative expression of HMGB1 in ANP group at 36 h was significantly higher than those at other time points (all P ＜ 0.05).HMGB1 protein expression in pancreatic tissue in ANP group at 6 h,18 h,36 h,72 h were 1.14 ±0.02,1.15 ±0.01,1.22 ±0.01,1.22 ±0.04,which obviously higher than those in control group(1.0),and HMGB1 expression in ANP group at 36 h was higher than those at other time points (all P ＜ 0.05).Conclusions HMGB1 may participate in systematic inflammation as one of the late inflammatory mediators during ANP.

Effect of different needle sizes and aspiration techniques on sample quantity / 中华消化内镜杂志

Objective To investigate the effect of needle sizes and aspiration techniques on sample quantity. Methods Aspiration was performed on porcine liver in vitro for 10 times with three different sizes of needles(19 G, 22 G and 25 G) and four different aspiration techniques[non?negative pressure(NP), 10 ml NP,20 ml NP and slow?pull], 20 mm in depth. A total of six aspirations were performed with each needle by the same aspiration technique. All the obtained specimens were fixed in formalin with the cell block method. The samples were evacuated according to our grading criteria. Results The mean±standard deviation(SD) score for 19 G,22 G, 25 G were 5?71±0?69,4?63±1?24, 3?79±1?84 respectively. The mean±SD score for methods non?NP,10 ml NP,20 ml NP and slow?pull were 4?72±1?53,4?56±1?46,4?72±1?50,4?83±1?76 respectively. The multi?analysis of variance results showed that there were statistical differences between different needles size( F=12?00,P<0?001) with 19 G being the best,followed by 22 G and the least specimen obtained by 25 G needle. There were no statistical differences among aspiration techniques ( F=0?128, P=0?943).The analysis showed that the thicker the needle was,the better sample quality was 19 G yielded to the highest quantity of specimens. The most specimens could be obtained with 19 G needle and non?NP, 22 G needle and 20 ml NP and 25 G needle and slow?pull. Conclusion In clinic, aspiration technique should be selected according to different aspiration needles. 19 G is superior to others, with non?NP method. For 22 G needle, 20 ml NP is preferred and for 25 G needle,slow?pull is preferred.

Liquid-based cytology diagnosis of endoscopic ultrasound-guided fine needle aspiration of pancreatic lesions / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the diagnostic value of liquid-based cytology test (LCT) in pancreatic lesions sampled by ultrasound-guided fine needle aspiration (EUS-FNA).</p><p><b>METHODS</b>A retrospective analysis of 556 cases of LCT smears sampled by EUS-FNA of pancreatic lesions was performed, and 164 cases had histologic diagnosis with subsequent surgical resection or biopsy and immunohistochemistry. The accuracy of the cytologic diagnosis was assessed using the histologic diagnosis as the gold standard. The discrepant cases were reviewed to identify sources of errors.</p><p><b>RESULTS</b>The satisfactory rate for EUS-FNA was 96.0%(534/556). The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy were 87.7%(128/146), 13/16, 97.7%(128/131), 41.9%(13/31) and 87.0%(141/162) respectively. The diagnostic accuracy was lower in cystic lesions than that in solid lesions. The LCT sensitivities of adenocarcinoma, lymphoma and neuroendocrine tumors were higher than those of cystic tumors and mesenchymal tumors. False positive diagnosis was mainly due to epithelial abnormalities in inflammatory reaction. False negative diagnosis was mainly due to scanty or lack of tumor cells in the smears, or mild atypia that was insufficient for diagnosis.</p><p><b>CONCLUSIONS</b>EUS-FNA is a valuable tool for the diagnosis of pancreatic lesions. Standardized terminology and nomenclature are helpful to improve the diagnostic accuracy.</p>

Identification of cut off value and lower limit of detection for K-ras mutation by PNA-PCR clamping method / 中华胰腺病杂志

Objective To determine the lower limit of detection (LLOD) and cut off values of K-ras mutation detection by peptide nucleic acid (PNA) clamping-PCR.Methods The genomic DNA of pancreatic cancer cell lines (PANC1 and SW1990) with codonl2,13 mutation and the genomic DNA of placenta with K-ras wild type were mixed and diluted serially into samples with different mutation rate (0,0.1％,0.2％,0.4％,0.8％,1.6％,3.1％,6.25％,12.5％,25％,50％),PANC1 cells with 1％ mutation rate and SW1990 cells with 30％ mutation rate and 4 samples with the quantity of DNA was 50,20,5,1 ng and 50,10,5,1 ng was prepared.Codon 12,13 mutation of K-ras was determined by PNA-PCR,and the mutation Ct values,overall Ct values were collected,and the △Ct values (mutation Ct values-overall Ct values) were calculated,and the tests were repeated for 10 times.ROC curve was used to analyze the △Ct values and determine the best cut off values for K-ras mutation,and the positive diagnostic rate,LLOD was evaluated.Results The mutation Ct,△Ct values of codon 12 mutation of PANC1 and codon 13 mutation of SW1990 of all the different mutation rates were statistically significantly different (P ＜ 0.05) when compared with negative control group,but the overall Ct values were not statistically significantly different from that of negative control group.For detection of K-ras codon 12 mutation by ROC curve,the relevant area of ROC curve (AUC) was 0.926,the optimum cut off value of △CT was 11,the sensitivity and specificity were 84％ and 100％,respectively,and the LLOD was 0.4 ng.For detection of K-ras codon 13 mutation by ROC curve,the relevant AUC was 0.906,the optimum cut off value of △CT was 9.5,the sensitivity and specificity were 71％ and 100％,respectively,and the LLOD was 1.5 ng.The mutation detection results of fixed rate further confirmed the LLOD.Conclusions This study successfully defines LLOD and cut off value of PNA clamping-PCR/K-ras method in detection of K-ras 12 and 13 codon mutations.This method meets the requirement of clinical application.

Expression of TAK1, pTAK1 proteins and density of tumor associated macrophages in pancreatic carcinoma / 中华胰腺病杂志

Objective To investigate the expression of TGF-β activated kinase1 (TAK1),phosphoryted TAK1 (p-TAK1) and the density of tumor associated macrophages (TAM) in human pancreatic carcinoma tissues and pairing adjacent normal pancreatic tissues,and explore their relationship.Methods The expression of TAK1,p-TAK1,CD68 (the marker of tumor associated macrophages) proteins in 57 samples of pancreatic cancer tissues and 35 samples of pairing adjacent normal pancreatic tissues were detected by immunohistochemical method,then the density of TAM was evaluated.The relationship between the protein expression and TAM,and the relationship between them and clinicopathologic parameters were examined using SPSS 18.0 software,and the independent risk factors of TNM staging were analyzed by multivariate logistic regression.Results TAK1 and p-TAK1 were positively expressed in 42.1％ (24/57) and 40.4％ (23/57) pancreatic carcinoma tissues,significantly higher than adjacent normal pancreatic tissues [14.3 ％ (5/35) and 11.4％ (4/35)];the proportion of pancreatic carcinoma tissues with high density TAM was 38.6％ (22/57),higher than that of adjacent pancreatic tissues [8.6％ (3/35)],the differences were statistically significant (P＜0.05).TAK1 expression was positively related to tumor size,tumor differentiation,lymph node metastasis,distant metastasis and clinical staging;p-TAK1 expression was positively related to tumor differentiation,lymph node metastasis,distant metastasis and clinical staging;the density of TAM was positively related tumor differentiation,lymph node metastasis,distant metastasis and clinical staging (all the P values were less than 0.05).The expression of TAK1 and p-TAK1were positively correlated with the density of TAM (P ＜0.001).Multivariate logistic regression analysis showed that high density of TAM was independently associated with advanced clinical staging (P =0.002,OR =129.5,95％ CI 6.2 ～2718.6).Conclusions TAK1 pathway and TAM may play an important role in the pathogenesis and progression of pancreatic cancer,and there may be synergy effect between them.

Effect of triptolide combined with gemcitabine on proliferation and apoptosis of pancreatic cancer cells / 重庆医学

Objective To observe the effect of triptolide combined with gemcitabine on proliferation and apoptosis of pancreatic cancer cells ,and to analyze the relevant mechanisms .Methods After treated with TPL ,GEM or TPL combined with GEM in vitro , PANC-1 cells proliferation was accessed by MTT assay and the interaction between the two drugs was calculated .Apoptotic mor-phological changes and apoptosis rate of the cells were investigated by Hoechst 33258 staining and flow cytometry ,respectively .The expression of signal transduction and transcription factor 3(STAT3) ,cysteine aspartate specific proteases-3(caspase-3) protein were detected by Western blot analysis .Results TPL ,GEM or TPL combined with GEM could significantly inhibit the prolifera-tion of PANC-1 cells ,and the combination of the two drugs had a synergistic effect .The cells of the TPL group ,GEM group ,as well as the combined group showed typical apoptotic morphological changes .Compared with the TPL group and GEM group ,the number of apoptotic cells of the combined group increased significantly .Compared with the control group ,the cells apoptosis rate of the TPL group ,GEM group and combined group was significantly increased (P< 0 .05) ,and the apoptosis rate of the the combined group was significantly higher than that of the monotherapy group(P<0 .05) .TPL combined with GEM synergistically inhibited p-STAT3 protein expression and activated caspase-3 protein expression .Conclusion TPL combined with GEM can synergistically in-hibit proliferation and induce apoptosis of pancreatic cancer PANC-1 cells ,its mechanism is related to the inhibition of STAT 3 sig-naling pathway ,promotion the expression of caspase-3 protein .

Expression and clinical significance of Shh, Gli1, Sufu, TAK1, p-TAK1 protein in pancreatic carcinoma / 中华胰腺病杂志

Objective To investigate the expression of Hedgehog signaling pathway members,Shh,Gli1,Sufu and TAK1 as well as phosphorylation-TAK1 (p-TAK1) protein in human pancreatic carcinoma tissues and to explore its relationship between the expression of proteins and the clinicopathologic parameters.Methods The expressions of Shh,Glil,Sufu,TAK1,p-TAK1 proteins in 38 samples of pancreatic cancer tissues and pairing adjacent normal pancreatic tissues were detected by immunohistochemical method.The relationship among the proteins and the relationship between the expression of the proteins and the clinicopathologic parameters were examined.Results The positive expression rates of Shh,Glil,Sufu,TAK1,p-TAK1 protein in human pancreatic carcinoma were 86.8％ (33/38),52.6％ (22/38),68.4％(26/38),55.3％ (21/38),52.6％ (20/38),but they were not expressed in adjacent normal pancreatic tissues.Gli1 expression was positively related to distant metastasis and clinical staging (r =0.524,0.361,P ＜0.05),Sufu expression was positively related to patient's gender (r =-0.378,P ＜0.05),TAK1 expression was positively related to clinical staging (r =0.468,P ＜ 0.05),p-TAK1 expression was positively related to clinical staging and distant metastasis (r =0.418,0.361,P ＜ 0.05).Gli1 expression was positively related to TAK1 and p-TAK1 expression in pancreatic cancer tissues (P ＜ 0.05).Conclusions Hedgehog signaling pathway and p-TAK1 may play a role in the pathogenesis of pancreatic cancer,and the two pathways may interact with each other.

A novel spliceosome encoding isoform of suppresser of fused in human pancreatic cancer / 中华胰腺病杂志

Objective To investigate spliceosome of suppresser of fused(SUFU),a major member of hedgehog signaling pathway in human pancreatic cancer.Methods SUFU fragment was amplified by using reverse transcription and 3' RACE.After sequencing,a new exon was discovered,and then nSUFU was amplified by RT-PCR.nSUFU and SUFU were transfected into SW1990 by liposomes,and then the expressions of SUFU protein encoded by new spliceosome in SW1990 cells and pancreatic cancer tissues were detected by Western blot.Results PCR products by 3'RACE were of 600 bp,after sequencing and comparison with Blast data of NCBI,it was detected that a new exon was inserted between SUFU mRNA isoforml (NM_016169.3) exon 10 and exon 11.After verification with SW1990,it was noted the entire new spliceosome containing new exon was of 1400 bp.SW1990 with nSUFU transfection strongly expressed nSUFU protein,and pancreatic cancer tissues expressed both SUFU and nSUFU protein.Conclusions A new spliceosome of SUFU,which can encode SUFU protein,is present in pancreatic cancer tissue and cell.

Construction and identification of RNAi eukaryotic expression vectors targeting human transcription factor glioma-associated oncogene homolog 1 / 中华胰腺病杂志

Objective To construct RNAi eukaryotic expressing vectors of human transcription factor glioma-associated oncogene homolog 1 (GLI1) with pGCsi-U6-GFP plasmid and to identify its activity in interfering GLI1.Methods Three GLI1siRNA targeting GLI1 were designed and synthesized according to the GLI1cDNA sequence in GeneBank,and then were cloned into pGCsi-U6-GFP to construct the recombinant plasmids,and transformed into E.coli DH5a,then it was amplified and plasmids were extracted,which were further confirmed by PCR reaction and DNA sequencing,pGCsi-U6-siRNA-C was negative as control wector.Then recombinant plasmids pGCs-U6-GLI1siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1siRNA-3 pGCsi-U6-siRNA-C and a eukaryotic over-expression vector pEGFP-N1-GLI1 were co-transfected into HEK293 cells by Lipofectamine 2000 respectively.The ceils were collected at 48 h after transfection.Semi-quantitative RTPCR and Western Blot were performed to detect the expression of GLI1 mRNA and protein to screen the optimal vector which had the best interfering effect.Results A 369 bp fragment was amplified from all three recombinant plasmids,(pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLIlsiRNA-3),showing that synthesized shRNA oligonucleotide fragments were correctly inserted into three recombinant plasmids,which were further confirmed by sequencing.Expression levels of GLIlmRNA and protein in cells in pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 were 0.290 ± 0.011,0.421 ± 0.018,0.373 ±0.018,and 0.318 ± 0.026,0.443 ± 0.021,0.381 ± 0.018,which were significantly lower than those in negative control group (0.834 ± 0.022,0.818 ± 0.024,P =0.000),the inhibitory rates were 65.8 ％,50.7％,55.7％,and 63.9％,48.3％,53.9％.The interfering efficacy of pGCs-U6-GLIlsiRNA-1 was the strongest among the three recombinant plasmids.Conclusions RNAi eukaryotic vectors pGCs-U6-GLIlsiRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 are successfully constructed and the optimal vector is identified,and this can provide a solid experimental foundation for further functional study of GLI1 gene.

Intratumor injection of recombined oncolytic adenovirus and ethanol in advanced pancreatic carcinoma combined with intravenous gemcitabine: a comparative study / 中华消化内镜杂志

Objective To compare the efficacy and safety of E1B gene-deleted adenovirus (H101)and ethanol in treating advanced pancreatic carcinomas by intratumoral injection combined with intravenous gemcitabine.Methods We constructed an orthotopic nude mouse model of pancreatic carcinoma through cancer cell injection into pancreas.A total of 54 nude mice were randomly allocated to 6 groups to accept H101,ethanol or saline (control) intratumoral injection,combined with or without intravenous gemcitabiein.The animals were sacrificed 4 weeks after the treatment and the pancreatic tumors were collected to determine the size,existence of metastasis,distribution of virus by indirect immunofluorescence and apoptosis in tumor by TUNEL and electron microscope.Results All mice completed the scheduled treatment,while 3 died in 48 hours after ethanol injection resulting in a mortality of 16.7％ (3/18).On the contrary,no mice died in the adenovirus injcction group.The average tumor size in group of H101 intratumoral injection combined with intravenous gemcitabie was significant smaller than that in group of saline injection with or without systemic gemcitabie (P =0.008,0.040,respectively).Similar differences were observed between ethanol intratumoral injection and control groups (P =0.012,0.041).Meanwhile,the H101 was absent in all the other organs except the pancreas,which meant that the selectivity of the H101 was tremcndous.The virus combine gemcitabie group had higher apoptosis rate in tumor (83.2 ± 35.7) ％,determined by TUNEL.Conclusion E1B gene-deleted adenovirus intratumral injection in combination with intravenous gemcitabine treating pancreatic carcinomas is efficient and safe,in spite of its lower effectiveness than ethanol.

Expression and role of DNMT3b in human pancreatic cancer tissue / 中华胰腺病杂志

Objective To investigate the expression of DNMT3b and its correlation with clinicopathological parameters of pancreatic cancer.Methods The expressions of DNMT3b protein in 12 pancreatic cancer tissues and para-cancerous tissues were detected by Western blot.The expressions of DNMT3b protein in 59 pancreatic cancer tissues were detected by immunohistochemistry.The association of DNMT3b expression and clinicopathological parameters of pancreatic carcinoma were analyzed.Results Western blot results showed the expressions of DNMT3b protein in pancreatic cancer tissues and para-cancerous tissues were 0.69 ±0.13and 0.14 ±0.03,and the protein level of DNMT3b in pancreatic cancer tissues were significantly higher than that in para-cancerous tissues (t =4.464,P ＜0.05 ).Immunohistochemistry examination showed that the positive rates of DNMT3b protein were 59％ in pancreatic cancer tissues and negative in para-cancerous tissues.DNMT3b expression was positively correlated with the TNM stage and lymph node metastasis.Conclusions DNMT3b is highly expressed in pancreatic cancer tissues,and it is related with malignant biological behaviors of pancreatic cancer cells.

Relationship between TLR4 expression and angiogenesis in human pancreatic adenocarcinoma / 中华胰腺病杂志

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.

Expression of β-catenin in PanINs， IPMNs and PDAC / 中华胰腺病杂志

ObjectiveTo detect the expressions of β-catenin protein in different pancreatic tissues ( NP,PanINs,IPMNs and PDAC) and evaluate its significance during the carcinogenesis of PDAC.Methods The expression of β-catenin protein in 12 samples of normal pancreatic tissues,52 samples of IPMN (IPMA 20 foci,IPMB 13 foci,IPMC 19 foci),PanINs 118 foci (PanIN-1 73 foci,PanIN-2 29 foci,PanIN-3 16 foci),50 cases of PDAC was determined by using immunohistochemistry. The correlation between β-catenin expression and clinicopathologic characteristics of PDAC was analyzed. Results β-catenin was mainly expressed in cell membrane of NP,the quantity was 4.38 ± 2.11 ; in PanINs,PDAC and IPMN,β-catenin membrane expression was significantly decreased or absent,the β-catenin membrane expressions of PanIN-1,PanIN-2,PanIN-3,PDAC,IPMA,IPMB,IPMC were 5.22 ±2.21,2.24 ±2.31,1.44 ±1.37,2.71 ±2.08,4.85 ±2.28,4.15 ±2.51,2.68 ±2.75.The cytoplasm expression of PanIN-1 was 12.3％ (9/73),while the nuclear expression was 0 ; and the corresponding values were 34.5％ (10/29) and 3.4％ ( 1/29 ) in PanIN-2; 43.8％ (7/16) and 12.5％ (2/16) in PanIN-3; 44.0％ (22/50) and 10.0％ (5/50) in PDAC.The IHCS of β-catenin membrane expression decreased with the severe tissue atypia along the progressive multistage.The β-catenin membrane expression was significantly associated with tumor size,neural infiltration and lymphatic metastasis ( P ＜ 0.05 ).Ectopic cytoplasm expression was significantly associated with tumor size (P＜0.05 ). Ectopicnuclear expression was significantly associated with tumor differentiation (P＜0.01).The membrane or ectopic cytoplasm expression of β-catenin was significantly associated with postoperative survival.ConclusionsAbnormal Wnt/β-catenin signal activation induces decreased β-catenin membrane expression and increased ectopic expression,which is an important mechanism of pathogenesis and development of PDAC,and promotes the growth and metastasis of PDAC.The expression of β-catenin was associated with postoperative survival.

Inhibition of the expression of inflammation cytokines by rhubarb in experimental acute necrotizing pancreatitis rats / 中华胰腺病杂志

Objective To observe the changes of inflammation cytokines in pancreas tissues of experimental acute necrotizing pancreatitis (ANP) rats treated with rhubarb.MethodsThirty-three rats were randomly divided into sham operation group,ANP group and rhubarb treatment group with 11 rats in each group.ANP model was induced by retrograde injection of 3％ sodium taurocholate into the biliopancreatic duct,and jejunostomy was performed.Rats in rhubarb treatment group were fed with 1 g/ml rhubarb at the dose of 1 ml/kg body weight via jejunum route.Thirty-six hours later the rats were sacrificed.The serum level of amylase was measured; part of the pancreatic tissue was harvested for routine pathologic examination; total RNA was extracted from pancreatic tissue.The expression of IL-6,IL-8,and TNF-α mRNA was measured by real-time PCR.ResultsAfter ANP induction,the serum level of amylase was significantly increased,and pancreatic tissue necrosis,bleeding and inflammatory cell infiltration were observed,which was consistent with changes of ANP.The expressions of IL-6,IL-8,and TNF-α mRNA were 0.29 ±0.13,0.35 ±0.15,1.09 ±0.32 in sham operation group,while they were 2.23 ±0.49,2.26 ±0.51,5.24 ±0.59 in ANP group,which were significantly higher than those in sham operation group ( P ＜ 0.05 ).The corresponding values were 0.97 ±0.30,1.02 ±0.34,2.59 ±0.36 in rhubarb treatment group,which were significantly lower than those in ANP group,but they were still significantly higher than those in sham operation group ( P ＜ 0.05 ).ConclusionsRhubarb lavage via jejunum route can decrease the expressions of IL-6,IL-8,and TNF-α,therefore attenuate the pancreatic pathologic injuries.

The effect of α-tocopherol on fibrosis of chronic pancreatitis rat / 中华胰腺病杂志

Objective To investigate the effect of α-tocopherol on fibrosis of chronic pancreatitis (CP) rat and explore its mechanism.MethodsMale Wistar rats were randomly divided into control group,acute necrotizing pancreatitis (ANP) group,α-tocopherol group.CP was induced by dibutyltindich loride ( 8 mg/kg) infusion into the tail vein.Gastric lavage of α-tocopherol (800 mg/kg body weight,daily) was started 24 hours after dibutyhindich loride infusion for 4 weeks.The rats in ANP and control group received 0.6 ml salad oil gastric lavage.The rats were sacrificed 4 weeks later.Pancreatic tissue was harvested for histological examination and collagen staining,and measurement of the levels of hydroxyproline and malondialdehyde (MDA) of the pancreas were performed.The mRNA expression of transforming growth factor (TGF)-β１ was measured by real time PCR.ResultsAfter gastric lavage for 4 weeks,the pancreatic tissue inflammation,fiber deposition and abnormal structure in rats of α-tocopherol group were greatly reduced.The levels of MDA and hydroxyproline in rats of α-tocopherol group were significantly lower than those in ANP group [ (0.40 ±0.20) vs (1.07 ±0.41) nmol/100mg,(402.49 ±27.62) vs (664.92 ±29.04) μg/g,P＜0.05].The expressions of TGF-β1 mRNA in rats of o-tocopherol group were significantly lower than those in ANP group (2.24 ± 0.89 vs 3.35 ± 0.66,P ＜ 0.05 ).Conclusions Tocopherol gamma can improve pancreatic inflammation and fibrosis by reducing the oxidative stress level and down-regulating the expression of TGFβ1mRNA in rats with CP.

Expressions of PPAR γ protein during the course of pancreatic fibrosis of chronic pancreatitis in Wistar rats / 中华胰腺病杂志

ObjectiveTo observe the expressions of PPAR-γ protein during the course of pancreatic fibrosis of chronic pancreatitis (CP) in Wistar rats and its significance.Methods Bibutyltin dichloride ( DBTC,8 mg/kg body weight) in a volume of 200 ml solvent was injected into the tail vein to establish the CP rat's model.Wistar rats were randomly divided into control group and 1,3,5,7,14,42 d group according to weights.Pancreatic tissue underwent routine pathological examination,and collagen accumulation in pancreatic sections was determined by staining for Sirius Red.Pancreatic myeloperoxidase (MPO)activity was determined.Expressions of α-SMA and PPAR-γ proteins were assessed by immunohistochemical method.Results Light microscopy showed signs of acute pancreatitis with interstitial edema and infiltration of neutrophilic granulocytes 7 d after DBTC injection.Acinar cells necrosis,atrophy,lymphocytes and monocytes infiltration,fibrosis within lobule and peri-lobule as well as pancreatic duct changes were found,which was in accord with the course from AP to CP.One days after induction,the activity of MPO,expressions of α-SMA was significantly increased[ (0.78 ±0.71) vs (0.15 ±0.05)U/g,6.67 ±3.14 vs 0,P＜0.05],then it did not increase with time of induction.Seven days after induction,collagen level reached the peak [ (45.42 ±15.99)％ ],which was significantly higher than that in control group [ (10.87 ± 2.28 )％,P ＜ 0.05 ].Collagen fibers accumulated from periductal to intra-acinar and/or inter-acinar areas.In control rats,the expression of PPAR-γ protein was positive only in vessel walls,and the expression level was 0.17±0.41 and increased with time of induction,then reached the peak of 4.83 ± 2.71 at 42 d.ConclusionsDuring the course of pancreatic fibrosis in rats,the expression of PPAR-γ protein is gradually increased,and plays a limited anti-inflammation and anti-fibrosis role.

Effects of histone deacetylase 1 gene silencing on cell cycle regulation of pancreatic cancer cell line PaTu8988 / 中华胰腺病杂志

Objective To investigate the effects of histone deacetylase 1 (HDAC1) gene silencing on cell cycle of pancreatic cancer cell line PaTu8988 and possible mechanism.Methods The PaTu8988 cells were routinely cultured and divided into control group,negative control siRNA group (c-siRNA),HDAC1 siRNA 15 nmol/L group and HDAC1 siRNA 30nmol/L group.After Liposomes 2000 transfection for 48 h,the Western blotting was used to detect the efficiency of HDAC1 gene silencing on protein levels and the expression of p21 protein.Cell cycle was evaluated by using flow cytometry.Results Compared with that of control group,the expression of HDAC1 protein in PaTu8988 cell of HDAC1 siRNA 30nmol/L group was significantly decreased,and the expression of p21 protein was significantly increased; the percentage of G2/M phase cells were significantly decreased[(21.48 ±3.67)％ vs.(28.28 ±2.94) ％,P＜0.05]; while the percentage of S phase cells were significantly increased[( 50.20 ± 6.85 ) ％ vs.( 32.49 ± 2.78 ) ％,P ＜ 0.05].Conclusions HDAC1 siRNA can efficiently and specifically inhibit the expression of HDAC1 in PaTu8988 cells,and can induce S phase cells arrest,which may be related with up-regulation of p21 protein expression.

Expression of receptor for advanced glycation end products in rats with acute necrotizing pancreatitis / 中华胰腺病杂志

Objectives To explore the expression of the receptor for advanced glycation end products (RAGE) in pancreas and the serum concentration of RAGE in rats with acute necrotizing pancreatitis (ANP).Methods Sixty four male Spraque-Dawley rats were randomly divided into control group,ANP 6,18,24,36,48,72,96 h group with 8 rats in each group.The rat model of ANP was established by injecting 20％ L-arginine intraperitoneally at the dose of 250mg/100g body weight for twice at the interval of 1 h.Rats in control group were injected intraperitoneally with the same amount of saline.The pancreas samples were histologically examined by light microscope and scored.The amount of ascites,levels of serum amylase and RAGE was determined; Real-time PCR was performed to detect the expression of RAGE mRNA in pancreatic tissue.Results Pancreatic injuries aggravated with time.The amount of ascites increased from ( 1.98 ± 0.64) ml at 6h to (8.69 ±0.62)ml at 96 h.Serum amylase level began to increase at 6h after L-arginine intraperitoneal injection and reached the peak value at 18 h[(5069.88 ± 603.25 ) U/L],and returned to normal at 36 h.The serum concentration of RAGE and RAGE mRNA expression in pancreatic tissue were ( 18.33 ± 2.99) ng/ml and 0.41 ±0.13 in the control group.The corresponding values increased at 6 h in ANP group,which were (30.31 ±5.03) ng/ml and 1.57 ±0.19,and they were significantly higher than those in the control group (P ＜0.05) ; they reached the peak value at 24 h[( 105.41 ±21.31 ) ng/ml and 4.23 ±0.73],which were significantly higher than those in other ANP groups ( P ＜ 0.05 ) ; at 96 h they decreased to the lowest point [( 33.54 ± 6.96) ng/ml and 1.19 ± 0.19],which were still significantly higher than those in the control group (P ＜ 0.05 ).Conclusions The expression levels of RAGE in pancreatic tissues and serum level of RAGE increase within 36 h of ANP onset,then decrease gradually,but they are always higher than normal values.

Construction of eukaryotic vector of human neuronal pentraxin 2 and establishment of stable transfected PANCI cell line / 中华胰腺病杂志

Objective To construct eukaryotie vector of human neronal pentraxin 2 (NPTX2),and obtain the sstable transfected PANC1 cell lines.Metods The full-length cDNA of NPTX2 was diget EcoRl and Notl,and was cloned into plasmid pcDNA3,1,which were confirmed by sequencing,then the PcDNA3,1(+)and pcDNA3,1(+)-NPTX2 vectors were transfected into PANC1 cells by LipofectamineTM 2000,The stable transfected PANC1 cell lines were selected by the abiliy of resistanc to G418.The NPTX2 mRNA expression of PANC1 in the selected clones was detected by real-time quantitative PCR.Results The eukaryotic vector pcDAN3,1(+)-NPTX2 was constructed successfully,stable transfected PANC1 cell line was established and confirmed by real-time quantitative PCR.Conclusions The construction of eukaryotic vector targeting NPTX2 and the established sstable transfected PANC1 cell line provided a solid experimental foundation for further studies on the function of NPTX2 in pancreatic cancer cells.