ObjectiveTo investigate the effects of 5-Aza-CdR on the transcriptional regulation through methylation of the DNA promoter protocadherin 8(PCDHg) gene in pancreatic cancer cell line Capan-2.The Capan-2 retardation in growth rate and apoptosis were assessed in when administered 5-Aza-CdR and the chemotherapy agent,gemcitabine.MethodsMTT and flow cytometry were used to analyze the cell growth inhibition and apoptosis when treated with 5-Aza-CdR or in combination with gemcitabine.Methylation-specific PCR,RT-PCR and western blot were performed to detect methylation state,mRNA and protein respectively of PCDH8 gene in 5-Aza-CdR-treated Capan-2cells.Results Capan-2 cells treated with 5-Aza-CdR showed a slower growth rate,and a significant growth inhibition when given both 5-Aza-CdR in combination with gemcitabine.Compared with single drug administration and control,5-Aza-CdR together with gemcitabine can induce a stronger apoptosis signal.Different concentrations 5-Aza-CdR of were able to reverse methylation,restore mRNA and protein levels of PCDH8 in Capan-2.Conclusion5 Aza-CdR may demethylate the PCDH8 gene,which would effectively remove the gene silencing caused by high methylation,and thus induce gene mRNA transcription and protein expression to inhibit cell growth and have collaborative antitumor functions with gemcitabine.

AIM: We hypothesized that PPAR? ligands stimulate endothelial-derived nitric oxide(NO) release to protect the vascular wall.Thus,the purpose of this study is to investigate the effects of ciglitazone(Cig) and fenofibrate(Fen) on angiotensin Ⅱ(AngⅡ)-induced decrease in endothelial NO synthase(eNOS) expression and NO production in human umbilical vein endothelial cells(HUVECs).METHODS: HUVECs were preincubated for 24 h with Cig(10~(-7), 10~(-6),10~(-5),10~(-4) mol/L) or Fen(10~(-5) and 10~(-4) mol/L),then incubated for 12 h with 10~(-7) mol/L AngⅡ.Total RNA was extracted,and the expression of mRNA and protein of eNOS was assessed by RT-PCR and Western blotting.NO production was measured by Griees method.RESULTS: In the presence of 10~(-7) mol/L AngⅡ for 12 h,NO production in cultured HUVECs was decreased(P

OBJECTIVE: To provide a reference about choosing the methods of isolating exosomes derived from tumor cells including laryngocarcinima Hep-2 cells by comparing advantages and defects of two methods of isolation and extraction exosomes. METHOD: Previously, laryngocarcinoma Hep-2 cells were cultivated massively, then the cells were processed with hot shock in 42 degrees C for 1 h. Sucrose density gradient centrifugation ultrafiltration (method 1): cells culture supernatant 90 ml was gathered, the supernatant was clarified through a 3/0.8 μm small filter to remove impurities and fragments which in larger diameter. Then the filtering fluid was concentrated and purified through sucrose density gradient centrifugation and ultrafiltration, the concentrated fluid was obtained. Exosome Isolation Kit (method 2): cells culture supernatant 4 ml was gathered, the solutions of the kit were added into the supernatant in proper sequence, then filtered by the special column, the concentrated fluid was obtained. Both products are observed by high resolution transmission electron microscopy. RESULT: Both methods could isolate and extract exosomes feasibly. In single high power view of transmission electron microscopy, exosomes of method 1 disperse better, but lower density, and more impurity in background, exosomes of method 2 arrange closer, higher density, and less impurity. CONCLUSION Exosome isolation Kit require less supernatant, cost less time, process procedure briefly, harvest higher yield. It may become a new option of isolating exosomes derived from Laryngocarcinoma Hep-2 cells.
Cell Line, Tumor ; Exosomes ; ultrastructure ; Humans ; Laryngeal Neoplasms ; pathology ; Microscopy, Electron, Transmission

Objective To examine the expression of metastasis-associated in colon cancer 1 (MACC1) gene in ovarian cancer cell lines and investigate its effect on biological behaviors of ovarian cancer cells.Methods The expression of MACC1 was examined by qRT-PCR and Western blot analysis in four ovarian cancer cell lines inculding OVCAR3,ES-2,SKOV3 and HO-8910.When the MACC1 was transfected to OVCAR3 cells,fluorogenic quantitative PCR was used to filter and identify MACC1 gene after the efficient silencing.Changes of adhesion in the cells were analyzed by an adhesion assay.Transwell migration and invasion assay and in vitro vascular mimicry assay were used to detect migration,invasion and angiogenesis of OVCAR3 cells in vitro.Results The expression of MACC1 gene was higher in OVCAR3 compared to other cell lines.qRT-PCR confirmed that the expression of MACC1 was silenced successfully after transient transfected MACC1-siRNA into OVCAR3 cells.After successful silencing the MACC1 expression,the adhesion ability was inhibited to some degree.In transwell migration assay,the numbers of cells in upper chamber passing through the membrane in transfected group were less than control groups (245.5 ±12.8,500.3±16.5 and 496.3±13.1 respectively),while in transwell invasion assay,the numbers of cells in upper chamber passing through the membrane in transfected group were less than the negative group and control group (185.3±14.1,405.7±9.1 and 416.3±11.5 respectively),both with markedly differences among the three groups.In tube formation assay,the distrubition of HUVECs was diffused with less junctions,and the average number of complete tubular structure was decreased in transfected group compared to the corresponding controls.Conclusion RNA interference inhibits the expression of MACC1 and effectively inhibits the metastasis and invasion abilities of ovarian cancer cells in vitro,and MACC1 is expected to become the target gene of ovarian cancer treatment.

Objective:To construct tumor cell model by determination of the pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid expressing steadily in mouse melanoma B16 cells.Methods:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid being constructed from the melanoma-associated antigen A3 genes sourcing laryngocarcinoma in advance was translated into the mouse melanoma B16 cells under the mediation of lipofectamine,and the positive clones were detected with G418.The expression of enhanced green fluorescent protein( EGFP) and MAGE-A3 mRNA in positive clones were detected by fluorescence microscopy and fluorescence quantitative PCR ( qRT-PCR ) assay, respectively.Results:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected into B16 cells successfully, the green fluorescence of fusion protein expression was found, and MAGE-A3 mRNA transcription in B16 cells expressions were detected in positive clones.Conclusion:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected effectively and expressed stably by liposome method in the B16 cells.The expression of MAGE-A3 tumor cell model has been successfully established,which provide data for the study of laryngocarcinoma immunotherapy.

Objective To construct replication deficient recombinant adenovirus of human monocyte chemoattractant protein-1(MCP-1) by homologous recombination.Methods The cDNA of MCP-1 gene was obtained from human liver tissue by using RT-PCR,and was subcloned into a transfer plasmid pAdTrack-CMV.The linearized recombinant transfer plasmid pAdTrack-CMV-MCP-1 was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral plasmid.The recombinant adenoviral plasmid was linearized and then transfected into HEK293 packing cells to produce virus particles.The recombinant adenovirus was detected by using PCR.Results The recombinant adenoviral plasmid was successfully established and confirmed by restriction endonuclease digestion.The expression of green fluorescent protein(GFP) was observed on the 5th day after transfection.The fragment of MCP1 gene was amplified by PCR.Conclusion The achievement of recombinant adenoviral plasmid and recombinant adenovirus of MCP-1 lay a foundation for further investigation of the function and application of MCP-1.

1)were up-regulated in test and 184 genes(RA1.5)and down-regulation(RA

AIM:To observe the influence of adenovirus latent infection on the oxidant/antioxidant balance in rat alveolar epithelial cells.METHODS:The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid pneo.GSH and MDA contents,the activities of major anti-oxidative enzymes including SOD,CAT,GPx,GST and ?-GCS were detected in oxidant stress.RESULTS:Adenovirus E1A expression repressed the activity of ?-GCS,and decreased GSH contents in oxidant stress.As a result,the activity of GPx and GST was decreased.The contents of MDA maintained high in oxidant stress.CONCLUSION:Adenovirus latent infection amplifies the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress,and adenovirus E1A protein decreases the activity of ?-GCS,which plays an important role in this process.

Objective To study the effect of BRMS1 on the invasion and metastasis of ovarian cancer cells. Methods BRMS1 small interfering RNA (BRMS1-siRNA) was transfected into human ovarian cancer cell-line OVCAR3 by liposome transfection method. The cells were divided into 3 groups: experimental group with BRMSl-siRNA, negative control group with siRNA that did not impact any gene, blank control group without any transfection. Changes of invasion and migration in the cells were per-formed using transwell invasion and migration assay. Results BRMS1 gene was silenced in OVCAR3 cell line successfully detecting by quantitative real-time RT-PCR and immunoblotting.In transwell invasion assay,the numbers of cells in lower chamber passing through the membrane in transfected group were more than negative control group and blank control group (190±8.5, 144±7.8 and 146±6.8 respectively) (t=8.747, t=8.869, P=0.000), while in transwell migration assay, the numbers of cells in lower chamber passing through the membrane in transfected group were more than negative control group and blank control group were (231 ±8.9, 177±9.7 and 182±7.9 respectively) (t=9.314, t=9.224, P=0.000), both with significant differences among the 3 groups. Conclusion BRMS1 gene could suppress the invasion and metastasis of ovarian cancer cells.

Objective To analyze the influence of adenovirus latent infection on gamma-glutamylcysteine systhetase(γ-GCS) in rat alveolar epithelial cells. Methods The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid. Glutathione(GSH) contents, the activity of γ-GCS were detected in oxidant stress. Then the leuel of protein expression, mRNA expression, and promoter transcriptional activity of glutamate-cysteine ligase catalytic subunit(GCLC) were further detected. Results GSH contents decreased because of adenovirus E1A expression in oxidant stress. E1A repressed the expression and activity of γ-GCS, messenger RNA expression, and promoter transcriptional activity of GCLC. Conclusion Adenovirus E1A decreased the activity of γ-GCS probably by repressed promoter transcriptional activity of GCLC. As a result, GSH contents were downregulated in oxidant stress. Thus Adenovirus latent infection amplified the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress, which may be an important mechanism of COPD.