Objective To study the role of glutamate receptor subtypes in nucleus tractus solitarius(NTS)in cardiac-somatic motor reflex (CMR)induced by intrapericardial administration of capsaicin,and to clarify the modulation mechanism of NTS to cardiac nociceptoion.Methods 60 SD rats were randomly divided into ibotenic (IBO)group, glutamate group, MK-801 group, MCGP group, MK-801 + MCPG group and DNQX group. The NTS microinjected with 130 mmol·L-1 IBO 100 nL,100,200,500 mmol·L-1 L-glutamate 100 nL,NMDA receptor antagonist 40 and 60 mmol · L-1 MK-801 100 nL, metabotropic glutamate receptors antagonist 25 and 50 mmol·L-1 MCPG 100 nL,25 mmol· L-1 MCPG 50 nL plus 40 mmol· L-1 MK-801 50 nL,non-NMDA receptor antagonist 20 and 50 mmol·L-1 DNQX 100 nL,respectively.The changes of CMR of the rats in various groups were observed.Results Compared with control group,the CMR of the rats in IBO group was decreased (P<0.05);the CMR was increased as the concentration increased in glutamate group(P<0.05);the CMR in MK-801 and MCPG groups were decreased (P<0.05);the CMR in MK-801+MCPG group was decreased (P<0.05);the CMR in DXQX group had no changes (P>0.05).Conclusion NTS play an facilictory role in cardiac nociception,and the NMDA receptors and mGluRs receptors mediate this facilitory modulation.

Objective To explore the role of metabotropic glutamate receptors (mGluRs)group Ⅲ and its subtypes mGluR7 and mGluR8 in nucleus tractus solitarius(NTS)in cardiac-somatic motor reflex (CMR),and to clarify the modulation role of mGluR Ⅲ and its subtypes in NTS in cardiac nociceptoion.Methods 40 SD rats were randomly divided into L-AP4 group,microinjection of mGluRs Ⅲ agonist L-AP4 0.1,1.0,10.0 or 20.0 nmol in NTS;AMN082 group,microinjection of mGluR7 agonist AMN082 1,2 or 4 nmol;DCPG group,microinjection of mGluR8 agonist DCPG 4, 6 or 8 nmol;MSOP group, microinjection of mGluR Ⅲ antagonist MSOP 20 or 100 nmol,20 nmol MSOP+410 nmol L-AP,20 nmol MSOP+2 nmol AMN082,20 nmol MSOP+6 nmol DCPG. The changes of CMR of the rats in various groups were observed.Results Compared with control,the CMR in L-AP4 and AMN082 groups was decreased (P<0.05);the CMR in DCPG group was increased (P<0.05);the CMR in MSOP group after injection of 20 nmol MSOP had no change (P>0.05);the CMR in MSOP group after injection of 100 nmol MSOP was increased (P<0.05);the CMR in MSOP group after injection of 20 nmol MSOP followed L-AP4 or AMNO82 had no change (P>0.05).Conclusion The group Ⅲ mGluRs in the NTS play an inhibitory role in cardiac nociception, and mGluR7 has anti-nociceptive effects while mGluR8 has pro-nociceptive effects.

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 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.

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 .

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.

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.

PurposeAlteration of the regional cerebral flow and cerebrovascular reserve (CVR) caused by cerebral artery stenosis or occlusion is an essential risk factor for ischemic stroke. This study aims to assess the CVR in patients with severe middle cerebral artery (MCA) stenosis or occlusion by using MR perfusion-weighted imaging (PWI) with CO2 inhalation stress test. Materials and MethodsPWI were performed before and after CO2 inhalation stress on 28 patients with severe middle cerebral artery stenosis or occlusion and 10 healthy volunteers. The regions of interest (ROI) were put on the affected hemisphere of the MCA blood supply area and the contralateral side in both groups. The relative cerebral blood volume (rCBV), relative mean transit time (rMTT), relative cerebral blood flow (rCBF) and CVR were measured.Results① The rCBV, rCBF and rMTT of the affected side were 149.16±33.01, 18.04±5.24, 8.65±1.81 before CO2 inhalation stress, and 156.23±21.60, 23.77±8.77, 8.72±3.01 after CO2 inhalation stress. The rCBV, rCBF and rMTT were signiifcantly increased (t=1.238, 2.561 and 2.647, P<0.05) after CO2 inhalation stress. The rCBV, rCBF and rMTT of the contralateral side were 176.22±40.12, 22.43±5.74, 8.10±3.71 before CO2 inhalation stress, and 198.54±39.87, 27.64±7.22, 8.03±2.97 after CO2 inhalation. The rCBV, rCBF was significantly increased (t=1.780 and 1.665,P<0.05) while rMTT was signiifcantly decreased (t=2.871,P<0.05) after CO2 inhalation.②The CVR of the affected side, contralateral side and control group were 18.9%, 26.8%, and 39.1%, respectively. There were significantly differences (t=1.604, 2.321 and 1.874,P<0.05) between the affected and contralateral side, the affected side and control group, the contralateral side and control group.ConclusionPWI with CO2 inhalation stress test is valuable in assessing the cerebrovascular reserve in patients with severe middle cerebral artery stenosis or occlusion.

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.