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 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 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 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 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.

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.

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.

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.

Objective To investigate the association of the LIPC-C480T (rs1800588) and lipid levels and dyslipidemia in different age-and-sex groups in Han Chinese population.Methods The serum TC,TG,LDL-C and HDL-C were detected by automatic biochemical analyzer in 2420 health adults (1527 men and 893 women).The genotypes of rs1800588 were detected by M ALDI-TOF MS.According to the age difference (≤44,45-59 and ≥60-year-old),the total samples were divided to young (241 men and 201 women),middle-aged (652 men and 360 women) and older (634 men and 332 women) groups.The effects of genotypes on 4 serum lipid indicators in each age-and-gender group were analyzed by one way analysis of variance (ANOVA),and the odd risk of genotypes on dyslipidemia was estimated by binary Logistic regression analysis.The P value less than 0.05 was considered statistically significant.Results The frequence of allele T for LIPC rs1800588 in this population is 39.4％.In each age group the lipid parameters are quite different between males and females.Compared with those with CC genotype,middle-aged and elder men with CT or TT genotype have higher TC and HDL-C levels,and elder men with TT genotype also have higher TC level ; young women bearing CT genotype have higher TC level,and the CT and TT genotypes have higher HDL-C levels,middle aged women with CT or TT genotype have higher TC and TG levels,and CT genotype also have higher HDL-C level,the elder women with TT genotype have higher HDL-C level.Compared with those CC genotype individuals,the risk for mixed hyperlipidemia and hypercholesterolemia increases 2.318 folds (P =0.004) and 2.571 folds (P ＜ 0.001) respectively,while the risk for low HDL-C decreases 1.908 folds (P =0.029) for TT genotypes individuals among elder males; the hypercholesterolemia risk increasc 1.688 (P =0.036) and 2.099 times (P =0.040) in CT and TT genotypes respectively,and the risks for hypertriglyceridemia and mixed hyperlipidemia are 2.060 (P =0.038) and 2.381 (P =0.019) times higher than those with CC genotype among middle-aged females.Conclusions The LIPC rs1800588 site associates with the lipid levels and dyslipidmia risk in Han Chinese in an age-and-sex model.This SNP site has higher impact on lipid levels and dyslipidemia among elder males and middle-aged females,and the T allele is the risk factor.