Objective To investigate the hemodynamics and structural effect of rat endothelial progenitor cells (EPC) transplant on pulmonary artery hypertension (PAH) induced by monocrotaline(MCT) in rats.Methods EPCs were identified and marked.Twenty-one days after injection of EPCs,the pulmonary hemodynamic parame- ters,average pulmonary artery pressure (mPAP),right heart index were determined.The vascular endothelial cells and pulmonary vascular structural changes were verified by fluoresccuse microscope.Results Compared with the model,EPCs treatment(n=10) decreased mPAP significantly (mPAP,EPCs:25.9?0.7 mmHg vs model group:29.3?2.2 mmHg,P

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 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.

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.

Objective To investigate the expression of GLI1 and PTCH1 mRNA in pancreatic cancer and study its clinical significance. Methods Real-time fluorescence quantitative PCR (RFQ-PCR) was used to detect the expression of GLI1 and PTCH1 mRNA in 35 samples of pancreatic cancer tissues and 27 samples of adjacent normal pancreatic tissues, and the correlation of GLI1 and PTCH1 mRNA expression with clinical parameters was investigated. Results The relative expression of GLI1 mRNA in pancreatic cancer tissues was 1.12 ～ 3. 65 ( median 1.19), the relative expression of TCH1 mRNA was 1.82 ～ 4.36 ( median 2.36 ). The relative expression of GLI1 mRNA in adjacent normal pancreatic tissues was 0.23 ～ 2.76 ( median 0.87 ), the relative expression of PTCH1 mRNA was 1.11 ～ 2. 17 (median 0.58). Both the expression of GLI1 and PTCH1 mRNA in pancreatic cancer tissues were significantly higher than those in normal pancreatic tissues (P<0.05), and a positive correlation was found between GLIl and PTCH1 mRNA expression levels (P <0.05 ). The expression of GLI1 mRNA was significantly correlated with the differentiation degree and lymph node metastasis of pancreatic cancer (P < 0. 05). Conclusions GLI1 and PTCH1 may be involved in pancreatic carcinogenesis, and GLI1 may be related to invasion and lymph node metastasis of pancreatic cancer.

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.

Objective To investigate the expression of SUFU protein in human pancreatic carcinoma tissues and to explore the relationship between the expression of SUFU protein and the clinicopathologic parameters.Methods The expression of SUFU protein in 28 samples of pancreatic cancer tissues and 20 adjacent normal pancreatic tissues and 4 normal pancreatic tissues was detected by immunohistochemical method.And the relationship between the expression of SUFU protein and the clinicopathologic parameters were determined.Results SUFU protein was positively expressed in cytoplasm and nucleus of pancreatic carcinoma cells, while it was not expressed in the duct, acinar and islet of tumor-adjacent tissues and normal pancreatic tissues, and difference was statistically significant (P ＜ 0.05).The high SUFU protein expression was related to the clinical stage ( P ＜ 0.05 ), but not the age, gender, tumor location , tumor size, lymph node metastasis and liver metastasis.Conclusions SUFU protein was highly expressed in pancreatic cancer, and SUFU may play certain role in the pathogenesis of pancreatic cancer.

Objective To investigate the effect of jejunal casein perfusion on pancreatic exocrine secretion in experimental acute necrotic pancreatitis (ANP) rats and analyze the neuromechanism that may be involved. Methods 30 SD rats were randomly divided into three groups (control group, ANP group and ANP jejunal nutrition group). Experimental ANP was induced by intra pancreatic duct injection of sodium taurocholate (STC). Animals in ANP jejunal nutrition group were given jejunal casein perfusion 24h after model induction, while control group and ANP group received jejunal saline perfusion. Pancreatic juice was collected every 15 min for six times and the volume of pancreatic juice and protein in pancreatic juice were detected. After jejunal nutrition c-Fos expression in nucleus tractus solitarii (NTS) was determined by immunohistochemistry method in three groups. Results There was no significant difference between the volume of pancreatic juice at different time points in ANP group and ANP jejunal nutrition group, however, these parameters were significantly lower than that of control group (P < 0. 05). There was no significant difference among the 3 groups in the protein level in the pancreatic juice during jejunal nutrition infusion, however, during the periods of 0 ～ 15 min, 15 ～30 min, 30 ～45 min and 75 ～90 min, the protein levels in the pancreatic juice in ANP and ANP jejunal nutrition group were lower than that of control group (P < 0.05). After jejunal perfusion, c-Fos expression was found in ANP jejunal nutrition group but not found in ANP and control groups. Conclusions Jejunal casein perfusion enhanced NTS c-Fos expression, but did not increase the volume of pancreatic juice and protein.

To improve the efficiency of halophilic actinobacteria screening and carry out the rapid selection of targeted strains, we tested 34 strains of Streptomonospora by PCR-single strand conformation polymorphism analysis (PCR-SSCP) based on genus-specific primers for the PCR identification. This approach employs PCR with two pairs of primers located in the 16S rRNA sequence flanking two variable region, then build clustering tree according as SSCP data. Synchronously, we sequenced all the 16S rRNA partial sequences for these strains to verify them. The results showed that the PCR-SSCP analysis was an efficient, easy-to-handle and economic method for rapid selection of halophilic actinobacteria resources.

Objective:To investigate the role of extracellular signal-regulated kinase 1/2(ERK1/2)signal pathway in the pathogenesis of stress-induced gastric ulcer.Methods:Animal model of stress-induced gastric ulcer was established in rats with water-immersion restraint(WIR)stress.The mucosal activation of ERK1/2 was observed before and 5,15 and 30 min,and 1, 2 and 3.5 h after WIR stress.Some animals were also treated with an intravenous injection of PD98059(1 mg/kg),a specific ERK1/2 inhibitor,1 h prior to WIR stress.Expression of total ERK1/2 and caspase-3 were detected by Western blot analysis; ERK1/2 activity was measured by kinase activity assay using myelin basic protein as a specific substrate.DNA-binding activities of the transcription factors activator protein-1(AP-1)and nuclear factor-?B(NF-?B)were determined by electrophoretic mobility shift assays(EMSA).Mucosal TNF-?and IL-1?mRNA expression was analyzed by Northern blot analysis.The degrees of the gastric mucosal lesions were expressed as ulcer index(UI)and pathological evaluation.Apoptosis in the gastric mucosa was examined by an in situ TdT-mediated dUTP nick-end labeling(TUNEL)method.Results:Activated ERK1/2 was very weakly expressed in the gastric mucosa of normal rats.ERK1/2 was rapidly activated in the gastric mucosa of rats 15 min after WIR stress and the activity reached the maximal after 3.5 h.Pretreatment with PD98059 significantly inhibited ERK1/2 activation,decreased AP-1 and NF-?B activities and TNF-?and IL-1?mRNA expression,and obviously relieved gastric mucosal lesions,accompanied by caspase-3 activation and increased apoptosis.Conclusion:The present results indicate that ERK1/2 activation plays an important role in the development of stress-induced gastric ulcer.