Objective To study the effect of the recombinant vaccinia virus expressing human interleukin 2 on MKN45 gastric cancer cells in vitro. Methods Recombinant vaccinia virus expressing hIL 2 (VMJ601hIL 2) was constructed by homologous recombination using molecular virology, and VMJ601hIL 2 was detected by DNA hybridization technique and the recombinant gene product was analyzed by SDS PAGE. In addition, MKN45 gastric cancer cells was infected by VMJ601hIL 2 and the effect of VMJ601hIL 2 on the gastric cancer cells was evaluated in vitro. Results hIL 2 protein with biological activity can be secreted by MKN45 gastric cancer cells after heavily infected by VMJ601hIL 2. Conclusions It is one of the crucial steps that VMJ601hIL 2 has been constructed and identified since it forms essential prerequisite for further in vivo gene therapy of gastric cancer.

Background:Tissue microarray has been increasingly used in research of malignancies. It has been revealed that TGF-β signaling pathway contributes to the tumorigenesis and progress of malignancies. Aims:To determine the expressions of Runx3,Smad4,Cdk2 and p21,the key molecules in TGF-β signaling pathway by tissue microarray,and investigate their correlations with clinicopathological features and prognosis of gastric cancer. Methods:A total of 378 paraffin embedded tissue blocks,including 130 gastric cancer tissue and 248 para-cancer tissue from 130 patients undergoing radical resection of gastric cancer were obtained. Tissue microarray and immunohistochemistry were used to determine the expressions of Runx3,Smad4,Cdk2 and p21. Results:The aberrant expression rates of Runx3,Smad4, Cdk21 and p21 in gastric cancer tissue were significantly higher than those in para-cancer tissue(67. 7% ,35. 4% , 63. 8% and 70. 0% vs. 14. 1% ,12. 5% ,18. 1% and 37. 1% ,P < 0. 05,respectively). Aberrant expression of Runx3 was closely correlated with histological grade and lymph node metastasis of gastric cancer( P < 0. 05),while aberrant expressions of Smad4 and p21 were correlated with histological grade only(P < 0. 05);aberrant expression of Cdk2 was correlated with histological grade,lymph node metastasis and TNM staging(P < 0. 05). Pairwise correlations were seen among aberrant expressions of Runx3,Smad4 and p21 in gastric cancer,while Cdk2 was correlated with Runx3 only. Kaplan-Meier survival curve showed that 5-year survival rates in Runx3,Smad4 and p21 aberrant expression groups were significantly lower than those in normal expression groups(P <0. 05). Furthermore,Cox proportional hazard model indicated that Runx3 and Smad4 were independent prognostic factors for gastric cancer. Conclusions:Runx3,Smad4,Cdk2 and p21 might play pivotal roles in tumorigenesis and progression of gastric cancer. As interactions occurred among these four proteins,whether Runx3 and Smad4 could be used for predicting the prognosis of gastric cancer needs to be further studied.

Objective:To prepare rabbit polyclonal antibody against human argonaute 3(AGO3) protein,to identify its properties and investigate the tissue distribution of AGO3 using tissue array.Methods:AGO3 peptide was synthesized using chemical method,and then conjugated to Keyhole limpet hemocyanin(KLH) as immunogen.The AGO3-KLH conjugate were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification using affinity chromatography.Then the distribution of AGO3 in tissues was examined through immuno-stainning by tissue array.Results:Rabbit polyclonal antibodies against AGO3 were raised after immunization with AGO3-KLH conjugates.The anti-serum titer after the last inoculation was up to 1∶20 000.The preparations of the antibody were confirmed to raised recognize AGO3 peptides specially by ELISA and Western blot.AGO3 protein was stained positively in the cytoplasm of tumor cells and epithelial cells in many normal tissues.Conclusion:The polyclonal antibody against AGO3 protein has been achieved successfully,and it provides an efficient tool for further studying the roles of AGO3 in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.

Objective To evaluate the impact of rosiglitazone (Ros) on liver expression of peroxisome proliferator-activated receptor γ (PPARγ),nuclear factor (NF-κB) and cyclooxygenase-2(COX-2) in treatment of nonalcoholic steatohepatitis (NASH) rats.Methods Thirty Sprague-Dawley rats were divided into normal group,model group and Ros treated group with 10 each.Except the normal group,the other two groups were given high fat diet for 12 weeks for NASH model.The rats in Ros treated group were gavaged 4 mg/kg of Ros daily at the 12th week for 8 weeks.All rats were sacrificed at the 20th week for blood sample and liver tissue.Biochemical parameters of liver function,lipid metabolism,glycometabolism and antioxidant enzyme activities were measured.The histological change of the liver were assessed with HE and Masson staining.The level of tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) was measured using ELISA.The expression of PPARγ,NF-κB and COX-2 was detected with immunohistochemistry.The mRNA and protein expressions of COX-2 were tested by real-time PCR and Western blotting,respectively.Results In comparison with model group,Ros treated group showed significant improvement in hepatic steatosis,inflammation and fibrosis(all P value<0.05).In model group,the serum levels of fasting blood glucose,insulin and HOMA-insulin resistance index (HOMA-IRI),total cholesterol (TC),total triglyeride (TG),lowdensity lipoprotein cholesterol (LDL-C) and free fatty acids were increased,but HDL-C level was decreased.All above parameters markedly improved after Ros treatment.The levels of ALT and AST,total anti-oxidation competence,superoxide dismutase,catalase,glutathione peroxidase and malondialdehyde in Ros treated group were significantly ameliorated when compared with those in model group.Immunohistochemistry showed that the expression of NF-κB and COX-2 was significantly elevated,but PPARγ was decreased in model group.Real-time PCR and Western blot revealed that the mRNA and protein expressions of COX-2 were higher in the model group than those in normal group (0.57±0.08 vs 0.38±0.03;2.83±0.24 vs 1.00±0.03,P=0.000 and P=0.004,respectively),but significantly lower in Ros treated group (0.55±0.06 and 1.84±0.13,P<0.01).Conclusions Ros can reduce oxidative stress and insulin resistance in NASH rats by activing PPARγ expression and inhibiting expression of NF-κB and cyclooxygenases.

Objective To prepare polyclonal antibodies against human PIWIL and to identify their property and tissue distribution of PIWIL.Methods PIWIL polypeptide was synthesized and conjugated to Keyhole limpet hemocyanin(KLH) as an immunogen.Then PIWIL-KLH conjugations were injected into rabbits subcutaneously to produce polyclonal antibodies.The specificity and sensitivity of antibodies were identified by ELISA and Western blot after purification by affinity chromatography.PIWIL were then stained on the tissue chip to study their distribution.Results Rabbit antibodies against PIWIL were prepared after injection of PIWIL-KLH conjugation.These antibodies specially recognized PIWIL peptides.Expression of PIWIL was found in the cytoplasm of epithelia cells of varied normal tissues and tumor tissues.Conclusion The successful preparation of the polyclonal antibody against PIWIL will provide an efficient reagent for further study of its role in the pathway of miRNA and RNA interference and in the pathogenesis of human disease.

Objective To investigate the gene expression difference of IFN and their receptors in peripheral blood mononuclear cells (PBMC) of pulmonary embolism (PE) patients.Methods Twenty cases of PE patients and twenty sex and age matched controls were recruited into the study.Human cDNA microarray analysis was used to detect the gene expression difference of IFN associated genes between the two groups,and random variance model corrected t test was used to analyze the statistical data.Results In comparison with the control group, mRNA expression of type Ⅰ IFN, including IFNα5 mRNA,IFNα6 mRNA,IFNα8 mRNA,IFNα14 mRNA,IFNκ mRNA,IFNω1 mRNA,IFNε1 mRNA in PBMC of PE patients Were down-regulated (P ＜ 0.05 ).There was no significant difference in gene expression of type Ⅰ IFN receptors IFNαR1 and IFNαR2 between the PE and control groups (P ＞ 0.05 ).In comparison with the control group,mRNA expression of IFNγ gene was down-regulated ( P ＜ 0.05 ).The mRNA expression of IFNγR1 and IFNγR2 genes were upregulated compared with the control (P ＞ 0.05 ).Conclusion mRNA expression of type Ⅰ and type Ⅱ IFN in PE are significantly down-regulated,but not the IFN receptors.Reduced immune function may play an important role in the PE patients who are susceptible to virus,intracellular bacteria and parasites.

Objective　To investigate the feasibility of applying transvescal approach laparoendoscopic single-site radical prostatectomy (TVSSLRP) and assess the oncological and functional outcomes.Methods　Eight patients with clinically localized prostate cancer (PCa) of low risk underwent TVSSLRP.Demographic data were accrued including patient age,body mass index (BMI),preoperative PSA level,the International Index of Erectile Function 5,biopsy Gleason score,clinical TNM stage and D'Amico risk classification.One surgeon performed all TVSSLRP procedures.A homemade triple-port was introduced percutaneouly into the bladder to establish pneumovesicum through a 4 cm incision.The major steps of the surgery were described as follows:initial incision was made along posterior margin of the bladder neck to expose bilateral vas deference and spermatic vesicle.After opening Denonvilliers' fascia and extending the space to lateral prostatic pedicles,an intra-fascial nerve sparing procedure was performed.The puboprastatic ligaments were then separated close to the prostate surface and the dorsal vein complex was cautiously swept off.Subsequently,careful apical dissection and urethral transection was sequentially conducted. To reduce the tension of vesico-urethral anastomosis,3 additional incisions parallel to vesio-urethral margin were created and a novel tension - reduced V-LocTM barbed polydioxanone sutures was used.　Results　All the operations were successfully performed and there was no conversion to standard laparoscopic approach or open surgery.The total operative time range was 75 - 180 min with mean time of 125 min.The blood loss was 85 -450 ml with mean 140 ml and no blood transfusion was required.The catheter was removed after a mean (range) of 14 (9 -16) days.No intra-operative complications occurred. No patient had positive surgical margins.The mean (range) hospital stay was 17 (13 -25) days after surgery. All the cases were continent after removal of the catheter.No cases demonstrated vesico-urethral stricture and biochemical recurrence on 12 - 18 months follow up postoperatively.　Conclusions　TVSSLRP is technically feasible for cases with organ-confined prostate cancer with good oncological and functional results.

Objective To study the expression of PIWIL1, PIWIL3 and PIWIL4 in human colon cancer and its clinical significance. Methods We collected cancerous tissues and its adjacent tissues of 106 patients with colon cancer, two tissue microarrays were constructed, with 62 and 150 points respectively. We studied the expression of PIWIL1, PIWIL3 and PIWIL4 through immunohistochemistry. Results The expression of PIWIL1, PIWIL3 and PIWIL4 were significantly higher in cancerous tissues than those in adjacent tissues (P＜0. 01). In cancerous tissues and its adjacent tissues, postive correlation were seen among PIWIL1, PIWIL3 and PIWIL4 expression (P＜0. 01). PIWIL1 expression was significant higher in low differentiation group than that in high differentiation group (t =- 2. 840, P＜0.01 ). PIWIL3 expression was higher in high clinical stage than that in low clinical stage (F= 3. 112, P＜0.05). The expression of PIWIL3 and PIWIL4 were significantly higher in patients with colon cancer with distant metastasis than those without distant metastasis (t= -3. 349, P＜0.01 ; t = - 2. 168, P＜0. 05). PIWIL3 and PIWIL4 expression were correlated with occurring of colon cancer (P＜0. 01). Conclusions The expressions of PIWIL1,PIWIL3 and PIWIL4 in colon cancer were correlated with the differentiation, clinical stage and distant metastasis of colon cancer. PIWIL3 and PIWIL4 expression were two independent related factors of occurring of colon cancer, which would be furtherly investigated to be served as novel markers for early diagnosis and promising molecular targets for colon cancer therapy.

Objectives To investigate the expression of mTOR signaling pathway in pancreatic cancer and export its signification. Methods 6 samples of pancreatic cancer and its paracancerous tissues specimens confirmed by surgery and pathologic examination were selected. RNA was extracted and expression profiles experiment was performed by using Agilent human whole genomic oligonucleotide microarrays. The expression of mTOR signaling pathway in pancreatic cancer was analyzed by bioinformatics. Results Totally 1276 differential gene were selected, and 691 were up-regulated in cancer tissue, while 585 were down-regulated.The highest score of KEGG pathway's Enrichment and gene count was hsa04150 in mTOR signaling pathway,with its Enrichment of 4.5622519 and gene count of 9, and the percentage of gene count was 1.15%, the EASE Score P value was 6.23 E-04, which had the most biological significance. Among those, there was significantly difference of expression of nine key genes including ULK2, PIK3R3, PDPK1, EIF4EBP1, PGF,VEGFB, ULK3, RICTOR and PIK3 R5 (P ＜ 0.05). Conclusions The pathogenesis of pancreatic cancer is related to the activation of the mTOR signaling pathway.

Objective To investigate the whole genomic expression profiles of chronic atrophic gastritis with interleukin(IL)-1β-31CC/-511TT genotype as measured by oligonucleotide microarray technique.Methods Genomic RNA was extracted from gastric biopsies of 12 patients with chronic atrophic gastritis(6 with IL-1β-31CC/-511TT and 6 with IL-1β-31TT/-511CC).The genomic profiles of IL-1β gene polymorphisms 31CC/-511TT and 31TT/-511CC were compared and tested for differential expressed genes associated with 31CC/-511TT using Agilent human whole genomic oligonucleotide microarrays.The results were further analyzed in terms of gene ontology(GO).Results There were 200 differentially expressed genes associated with IL-1β-31CC/-511TT,159 of which were up-regulated and 41 were down-regulated.These genes mainly involved in macromolecule metabolic process,post-translational protein modification,ubiquitin cycle,and protein kinase cascade.Five genes had biological activities,one of which was down-regulated gene(PCSK5)and 4 were upregulated genes(PRKCA,NPLOC4,TRIB3 and MAPKAPK3).Conclusions The chronic atrophic gastritis with IL-1β-31CC/-511TT genotype has molecular phenotypes which is associated with malignance and inflammation.These individuals are needed more intensive preemptive treatment and dynamic surveillance.