Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge in oncology due to its dismal prognosis and limited therapeutic options. In this study, we investigated the role of miR-301a in facilitating crosstalk between the Hedgehog (Hh) and HIPPO/YAP signaling pathways during the progression of PDAC. Our findings revealed that miR-301a served as a central regulatory node, targeting Gli1 within the Hh pathway and STK4 within the HIPPO/YAP pathway. Immunohistochemical and molecular analyses confirmed dysregulation of pathway components in pancreatic cancer, underscoring the pivotal role of miR-301a. Functional assays demonstrated the impact of miR-301a on cell proliferation and apoptosis, particularly in synergy with TNF-α. Overall, our study elucidated the intricate interplay between the Hh and HIPPO/YAP pathways mediated by miR-301a, providing valuable insights into potential therapeutic strategies for intervening in PDAC.

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.

Objective: Post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) is the most common complication following ERCP and the mechanism is not fully understood. This study evaluated the changes in the inflammatory response, cellular apoptosis, and tight junction integrity in a rat model of pancreatitis to explore the underlying mechanism. Methods: PEP was induced in rats by retrograde biliopancreatic ductal infusion of contrast agents or saline. Pancreatic tissues were harvested and evaluated by histopathologic, immunohistochemical, immunofluorescence, and Western blot analyses. In addition, amylase and proinflammatory cytokines in plasma were quantified by ELISA assay. Results: PEP rats developed more severe acute pancreatitis than the sham group after injection of the contrast agent or isotonic saline. PEP rats exhibited increased tissue damage, plasma amylase, proinflammatory cytokines, necrosis, inflammatory infiltrates, apoptosis, and tight junction disruption. At the molecular level, contrast agent and isotonic saline-injected PEP rats demonstrated elevated NF-kappa B p65 and STAT3 pathways activation, altered expression and activation of apoptosis-related proteins, and suppressed expression of tight junction molecules. However, the contrast agent concentration had no effect on these changes. Conclusions: In models of acute pancreatitis induced using contrast agent and hydrostatic pressure, the contrast agent and high hydrostatic pressure easily induced the inflammatory response, apoptosis, and tight junction disruption. It is noteworthy that no significant difference in damaged pancreatic acinar cells was observed with different concentrations of the contrast agent. (C) 2021 IAP and EPC. Published by Elsevier B.V. All rights reserved.

BACKGROUND:Inflammation, apoptosis and oxidative stress contribute to the development of severe acute pancreatitis-associated acute lung injury (SAP-ALI). Currently, there is no curative treatment for SAP-ALI in the clinic. This study investigated the potential therapeutic role and mechanisms of colchicine in a rat model of SAP-ALI. METHODS:Male Sprague-Dawley rats were randomized and administrated intragastrically with vehicle saline or colchicine (0.5 mg/kg/day) for seven days, followed by injecting sodium taurocholate to induce SAP-ALI. Together with a healthy control group of rats, their pancreatic and lung tissues and plasma samples were collected for histology, enzyme-linked immunosorbent assay (ELISA), immunoblot, immunohistochemistry, and immunofluorescence. RESULTS:Compared with the sham controls, the SAP group of rats with vehicle saline treatment displayed severe damages, inflammation with many neutrophil and macrophage infiltrates in pancreatic and lung tissues, accompanied by elevated levels of plasma interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF)-α, which were significantly mitigated in colchicine-treated SAP + COL group of rats. Furthermore, colchicine treatment significantly attenuated nuclear factor kappa-B (NF-κB)-p65, signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) phosphorylation, reduced inducible nitric oxide synthase (iNOS) and 4-Hydroxynonenal expression, ROS production and cell apoptosis by decreasing caspase-3 cleavage, Bax expression, but increasing Bcl-2, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in pancreatic and lung tissues, relative to the SAP group of rats. CONCLUSION:Colchicine treatment significantly mitigated the severity of SAP-ALI by inhibiting inflammation, oxidative stress and cell apoptosis in rats.

Objective To explore the effects of ApoC3 gene on the severity of hypertriglyceridemiainduced acute pancreatitis (AP).Methods ApoC3 transgenetic mice and C57BL/6J mice AP model was induced by cerulein intraperitoneal injection,and ApoC3 transgenetic mice and C57BL/6J mice injected by normal saline solution in equal volume served as control group.Serum triglyceride and cholesterol were detected,and the pathological changes of the pancreas were observed.RT PCR method was used to examine the changes of the inflammatory factor including IL-1β,IL-6,α-SMA and TNF-α mRNA levels,which reflected the severity of the inflammation.Results Serum triglyceride and cholesterol were higher in ApoC3 transgenetic mice than in C57BL/6J mice [(3.434 ± 0.931) mmol/L vs (0.766 ± 0.120) mmol/L,(2.553 ±0.178) mmol/L vs (1.996 ± 0.080) mmol/L],and the differences were statistically different (P ＜ 0.05).The pathological changes of the pancreas were more severe in ApoC3 transgenetic AP mice than in C57BL/6J AP mice,and the IL-1β,IL-6 and α-SMA mRNA levels in the pancreatic tissue were obviously higher in ApoC3 transgenetic AP mice than in C57BL/6J mice (1.72 ± 0.07vs 0.78 ± 0.09,1.58 ± 0.09vs 0.87 ±0.04,0.83 ± 0.05vs 0.44 ± 0.04),and the differences were statistically significant (P ＜ 0.05),while there was no statistical difference on TNF-αmRNA level (0.70 ± 0.09vs 0.65 ± 0.08,P ＞ 0.05).Conclusions ApoC3 gene could aggravate the severity of the inflammation in hypertriglyceridemia-induced AP.

The objective of the present study was to construct an alginate (AG)-based phase-changeable and injectable hydrogel for imaging-guided tumor hyperthermia and chemotherapy. Based on the binding between the α-l-guluronic blocks of AG and calcium ions, the AG/MoS2/Bi2S3-poly(ethylene glycol) (MBP)/doxorubicin (DOX) solution formed a cross-linked hydrogel to simultaneously encapsulate MBP nanosheets and DOX within the hydrogel matrix. The in situ formed hydrogel can act as a reservoir to control the release of entrapped drug molecules, and the doped MBP nanosheets and DOX can realize computed tomography/photoacoustic dual-modal imaging-guided in vivo tumor photothermal therapy and chemotherapy, respectively. The AG/MBP/DOX hydrogel exhibited excellent photothermal conversion properties with mass extinction coefficient of 45.1 L/g/cm and photothermal conversion efficiency of 42.7%. Besides, the heat from the photothermal transformation of MBP can promote drug diffusion from the hydrogel to realize on-demand drug release. Additionally, the hydrogel system can restrain MBP and DOX from entering into the blood stream during therapy, and therefore substantially decrease their side effects on normal organs. More importantly, the drug loading of the AG hydrogel was general and can be extended to the encapsulation of antibiotics, such as amoxicillin, for the prevention of postoperative infections.

Objectives: The aim of this study was to determine the prognostic value of YKL-40 expression in patients undergoing curative resection of pancreatic cancer.Methods: This cohort study included 234 consecutive patients with pancreatic ductal adenocarcinoma who underwent curative resection. Surgical specimens were immunohistochemically assessed for YKL-40 expression. Kaplan-Meier method and Cox regression were used to evaluate the prognostic impact of YKL-40 expression. A multivariate logistic regression model was performed to examine the correlation between YKL-40 expression and tumor stage.Results: Of the 234 patients, YKL-40 overexpression was detected in 149 (63.7%) patients. Survival curves showed that patients with YKL-40 overexpression had significantly shorter survival time than those with low YKL-40 expression (P < 0.001). Cox regression analysis indicated that YKL-40 expression was an independent prognostic factor for both overall survival (hazard ratio, 3.82; 95% confidence interval [CI], 2.38-6.13) and progression-free survival (hazard ratio, 3.73; 95% CI, 2.33-5.99). Multivariate logistic regression analysis demonstrated that YKL-40 overexpression was an independent predictor for advanced tumor stage (odds ratio 4.15; 95% CI, 1.35-12.71).Conclusions: YKL-40 overexpression predicts poor prognosis and advanced tumor stage in patients undergoing curative resection of pancreatic cancer. Application of adjuvant treatment targeting the YKL-40 pathway may improve prognosis.

目的 探讨Toll样受体-4(TLR4)和p38丝裂原活化蛋白激酶(p38 MAPK)信号通路在胰腺癌血管生成中的作用.方法 以脂多糖(LPS)、TLR4-siRNA及p38 MAPK信号通路阻断剂SB203580分别作用于体外培养的胰腺癌PANC-1细胞,Western blot检测TLR4、血管内皮生长因子(VEGF)、磷酸化p38丝裂原活化蛋白激酶(p-p38)蛋白表达.收集各种因素处理后的PANC-1细胞培养液,观察其对人脐静脉内皮细胞(HUVECs)增殖、迁移和管腔形成的影响.结果 LPS组的HUVECs增殖率、迁移数目和管腔形成个数分别为(139.2±12.6)％、48.1±9.1和47.8±9.6,均显著高于对照组(P＜0.05).TLR4-siRNA组的HUVECs增殖率、迁移数目和管腔形成个数分别为(60.2±8.7)％、31.3±4.5和17.2±3.3,均显著低于对照组(P＜0.01);SB203580组的HUVECs增殖率[(79.6±8.9)％]、迁移数目(21.6±4.3)和管腔形成个数(23.5±4.3)均较对照组显著减少(P＜0.05);且TLR4-siRNA+ LPS、B203580+LPS组的HUVECs增殖率、迁移数目和管腔形成个数均分别显著低于LPS组(P＜0.01).与对照组相比,LPS组VEGF、p-p38蛋白表达均明显增加,TLR4-siRNA组、SB203580组TLR4、VEGF、p-p38蛋白表达明显减少;且TLR4-siRNA+ LPS组、SB203580+LPS组VEGF、p-p38表达较LPS组明显减少.结论 TLR4可促进胰腺癌的血管生成,其机制与激活p38 MAPK信号通路、促进VEGF表达有关.

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.

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.

Deregulation of Toll-like receptor 4 (TLR4) is closely associated with the progression of various types of cancers, but its role in pancreatic carcinogenesis is unclear. This study aimed to investigate the role of TLR4 in the angiogenesis of pancreatic cancer and the underlying molecular mechanisms. The culture supernatant (conditioned medium) of PANC-1 cells after appropriate treatment was used for the treatment of HUVECs. The proliferation, migration and tube formation of HUVECs were assessed by MTT, Transwell and Matrigel, respectively. In pancreatic cancer tissues, TLR4, VEGF and CD31 were upregulated as determined by immunohistochemistry and the expression of TLR4 and VEGF was positively correlated with microvessel density as detected by CD31 staining. Activation of TLR4 signaling by LPS in PANC-1 cells resulted in increased VEGF and phosphorylation of AKT, which were abolished by TLR4 silencing with siRNA and PI3K/AKT signaling inhibitor LY294002. The conditioned medium from PANC-1 cells treated with LY294002 or transfected with TRL4 siRNA reduced the proliferation, migration and tube formation of HUVECs. In contrast, the conditioned medium from PANC-1 cells treated with LPS stimulated the proliferation, migration and tube formation of HUVECs, which was however significantly inhibited by pretreatment of PANC-1 cells with LY294002 or transfection with TRL4 siRNA. Our findings suggest TLR4 may promote angiogenesis in pancreatic cancer by activating the PI3K/AKT signaling pathway to induce VEGF expression.

Objective To culture C57BL/6 mouse pancreatic stellate cells (PSCs) with a modified method,and to establish a simple cultivation method with a high yield of PSCs.Methods Pancreatic tissues in C57BL/6 mice were collected,washed by fetal bovine serum,minced into 0.5 ～ 1 mm3,and cultured in sterile culture flasks.After 4days,primary PSCs were collected and passed on.The changes in intracellular lipid droplets were identified by oil red O staining;α-SMA,desmin expression was detected by immunocytochemical or immunocytofluorescent staining,and Western blot.Results Outgrowth cells in the fourth day were oval or star with abundant lipid droplets and fewer cells expressed c-SMA and desmin protein.PSCs turned into bigger,more pseudopodial ones with decreasing lipid droplets and significantly increasing expression of o-SMA and desmin protein after the passage.Protein expression of α-SMA in primary and passage PSCs were 0.653 ± 0.071,2.290 ± 0.055,respectively;and the expression in passage PSCs was significantly higher than that in primary PSCs (P ＜0.05),and the number of cells expressing desmin protein was significantly increased.Conclusions Modified tissue culture is a high yield and high purity method for both quiescent and activated PSCs,which can meet the need of in-vitro studies.

Objective To investigate the effect of Gsh2 silencing on the sonic hedgehog (Shh) and GLI1 gene expression,which are the key factors of hedgehog pathway in human pancreatic cancer cell SW1990.Methods Three shRNAs tagerting Gsh2 (Gsh2-shRNA) were constructed and were inserted into pLKO.1 puro vector by double digestion with restriction enzyme,the empty vector without shRNA was used as a negative control.After plasmid amplification,plasmids DNA was extracted,and underwent electrophoretic separation and sequencing by double digestion.After confirmation by gene sequencing,plasmid was transfected into the human pancreatic cancer cell line SW1990,then real time PCR was used to detect the Gsh2 mRNA expression,and the plasmid with best silencing effect was selected to transfect the SW1990 cells,real time PCR and Western blot were respectively used to detect the mRNA and protein expression of the key factor in hedgehog pathway(Gsh2,Shh,Gli1).Results The gene sequencing of the 3 recombinant plasmids showed that Gsh2-shRNA were correctly constructed.If the mRNA expression of empty vector transfection cells was 1,the Gsh 2 mRNA expressions of SW1990 cells which were transfected by recombinant Gsh2-shRNA-1,Gsh2shRNA-2,Gsh2-shRNA-3 were 0.41 ± 0.02,0.22 ± 0.043,0.53 ± 0.03,and Gsh2-shRNA-2 had the best silencing effect.If the mRNA or protein expression of empty vector transfection cells was 1,the Gsh2,Shh,Gli1 mRNA expressions of cells with Gsh2-shRNA-2 transfection were 0.12 ±0.02,0.97± 0.13,0.19± 0.03,and the protein expressions were 0.55 ± 0.12,0.74 ± 0.06,0.53 ± 0.09.The Gsh2,Glil mRNA and protein expressions with Gsh2-shRNA-2 transfection were significantly down-regulated when compared with those of empty vector transfection cells,and the difference between the two groups was statistically significant (P＜0.01 or ＜0.05).Conclusions Gsh2 gene silence in pancreatic cancer cell line SW1990 can repress the expression of GLI1 in hedgehog pathway without affecting Shh expression.

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.

Objective To investigate the effect of TNF-α and IL-1β on the expression of human pancreatic cancer PaTu8988 NF-κB and Hedgehog (HH) signaling pathways members Gli1,Shh,SMO,SuFu.Methods Pancreatic cancer cell line PaTu8988 was treated with TNF-α and IL-1β,respectively,for 48 h,and cells without treatment were control group.Then the protein and mRNA expression of NF-κB and Shh,SMO,Gli1,SuFu was determined by RT-PCR and Western blot; apoptosis of cells were tested by flow cytometry.Results The expressions of NF-κB mRNA in PaTu8988 cells in TNF-α group,IL-1β group,control group were 9.92 ±0.78,7.74±0.32,1.01 ±0.08; and the expressions of Gli1 mRNA were 7.25 ± 0.45,5.74 ± 0.33,1.00 ± 0.06 ; and the expressions of Shh mRNA were 3.60 ± 0.36,4.33 ± 0.45,1.00±0.04; and the expressions of SMO mRNA were 1.03 ±0.15,1.07 ±0.16,1.01 ±0.06; and the expressions of SuFu mRNA were 0.88 ±0.14,0.96 ±0.13,1.01 ±0.05.The expressions of NF-κB,Gli1,Shh mRNA in TNF-α group,IL-β group were significantly higher than that in control group,and the difference between the two groups was statistically significant (P ＜ 0.05).The expressions of corresponding proteins were consistent with the expressions of mRNA.The apoptosis rates in TNF-α group,IL-1β group,control group were (17.40±2.87)％,(11.05 ± 1.34)％,(49.90±2.96)％,and the apoptosis rates in TNF-α group,IL-1β group were significantly lower than that in control group,and the difference between the two groups was statistically significant (P ＜0.01).Conclusions TNF-α and IL-1β can activate the expression of NF-κB and Shh,Gli1 in human pancreatic cancer PaTu898,and decrease apoptosis.

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.

Increasing evidence has confirmed the existence of cancer stem cells (CSCs) in both hematological malignancies and solid tumors. However, the origin of CSCs is still uncertain, and few agents have been capable of eliminating CSCs till now. The aim of this study was to investigate whether bulk pancreatic cancer cells could convert into CSCs under certain conditions and explore whether metformin and curcumin can kill pancreatic CSCs. Aspc1, Bxpc3 and Panc1 pancreatic cancer cells were cultured in stem cell culture medium (serum-free Dulbecco's modified Eagle medium/Nutrient Mixture F-12 containing basic fibroblast growth factor, epidermal growth factor, B27 and insulin) for 5 days and it was found that all the pancreatic cancer cells aggregated into spheres and expressed pancreatic cancer stem cell surface markers. Then characteristics of Panc1 sphere cells were analyzed and cytotoxicity assays were performed. The results show that Panc1 sphere cells exhibited CSC characteristics and were more resistant to conventional chemotherapy and more sensitive to metformin and curcumin than their parent cells. These findings suggested that bulk pancreatic cancer cells could acquire CSC characteristics under certain conditions, which may support the "yin-yang" model of CSCs (interconversion between bulk cancer cells and CSCs). These results also showed that metformin and curcumin could be candidate drugs for targeting pancreatic CSCs.

目的 比较3种血细胞DNA提取方法(进口试剂盒、国产试剂盒和改良苯酚抽提方法)对新鲜血细胞及冻血细胞DNA的提取效率及差异.方法 收集20例胰腺癌患者5 mL外周血标本,经低速离心获得血细胞沉淀后分装,根据保存方式分为新鲜血细胞和冻血细胞.新鲜血细胞不经冻存,在12 h内迅速抽提DNA;冻血细胞在-40℃保存72 h后,室温解冻后抽提DNA.抽提方法分别采用进口试剂盒(Qiagen公司)、国产试剂盒(天根生化科技有限公司)和改良苯酚方法.琼脂糖电泳检测DNA片段的完整性,核酸蛋白检测仪测定DNA纯度和浓度,计算不同抽提方法的DNA得率.结果 在获得的DNA完整性方面,改良苯酚方法抽提的基因组DNA断裂和降解较少,而进口和国产试剂盒抽提的DNA均有一定程度的降解小片段.在获得DNA的纯度方面,部分冻存血细胞无论应用进口试剂盒、国产试剂盒和改良苯酚方法,均表现出一定的污染杂峰.在DNA得率方面,无论新鲜血细胞和冻存血细胞,均是进口试剂盒得率最高,其次为国产试剂盒;冻血细胞的改良苯酚法DNA得率与国产试剂盒相当,新鲜血细胞的改良苯酚法DNA得率最低.在耗时和成本分析中,试剂盒较快但成本较高.结论 改良苯酚抽提法可获得较进口和国产试剂盒片段更长、更完整的基因组DNA,虽然在得率和耗时方面稍逊色,但其成本较低,可推荐用于大规模、分批次、低成本冻存血细胞的基因组DNA抽提.