Objective To explore the upstream signal transduction pathway of neutrophil elastase(NE)-induced mucin(MUC)5AC gene expression.Methods A549 cells were either incubated with NE alone or with DMTU,aprotinin or AG1478.The level of MUC5AC mRNA was measured with RT-PCR.The activation of epidermal growth factor receptor(EGFR) and signaling were assessed by measuring the release of epidermal growth factor(EGF) and the phosphorylation of EGFR.Results NE increased MUC5AC gene expression accompanied by an increase of EGF and phosphorylated EGFR.DMTU,aprotinin and AG1478 significantly inhibited MUC5AC gene expression.DMTU and aprotinin significantly decreased the level of EGF and phosphorylated EGFR.ConclusionNE can induce MUC5AC gene expression via EGFR signalling in A549 cells.The upper stream involves oxidants,activation of tissue kallikrein and EGF.

OBJECTIVE To observe whether the natural antibacterial polypeptide elafin after transfected into airway epithelial cells(A549 cells)has resistant effects on Pseudomonas aeruginosa bioflim.METHODS To establish the P.aeruginosa biofilm model in vitro,a rapid silver nitrate staining procedure was used to demonstrate bacterial biofilm.After cultivating the cells in vitro,the constructed pEGF-N1-elafin eukaryotic expression vector had transfected into A549 cells by LipofectamineTM 2000.Elafin transfected cells were incubated by pig Pancreas Elastase(NE group),the supernatants of PAE(PAE group)and Escherichia coli(ECO group),respectively,for 24 hours.Then the content of elafin in cells and the level of elafin secretion were detected by Western blot and ELISA respectively.After biofilm carriers were put into each group and incubated for 8 hours,we observed the ratio of cells shape breakage at 4,8,12 and 24 hour,respectively.RESULTS The NE,PAE and ECO groups induced the content of elafin in cells and the level of secretion increased compared to the no induced group(normal group),while the PAE group and NE group were higher than those of ECO group.The ratio of cells breakage in induced elafin groups was lower than that of in normal group(P

The method for examining the microcirculation bloodflow of mice mesentery was used to evaluate the action of Shujinhuoxuedingtong wine against microcirculation disturbance caused by adrenaline. The result indicates that the wine can obviously improve the microcirculation function and is responsible for the antistatic action observed. The wine's effect is found to be superior to that of Shujinhuoxuedingtong Powder. The above fingings suggest that the wine have the action of promoting blood circulation to remove blood stasis and improving microcirculation and the effect of promoting the bloodflow perfusion quantity in tissue organs. It's effect is prior to that of powder.

Objective To explore the molecule mechanism of protective effects of transfecting human elafin gene recombinant plasmid to NCI-H292.Methods Constructe eukaryotic expression vector pEGFP-C1-elafin and transfectied it into NCI-H292 cells,then co-incubated with polymorphonuclear granulocyte(PMN) with stimulation by lipopolysaccharide(LPS).Afater 24 hour,Western bolt and MTT technique were respectively performed to detect expression of ZO-1 in cells and adhesion capability of cells with collagen.Results After stimulation with LPS,Western blot showed that the 220 ku onula occludens-1(ZO-1) protein in transfecting pEGFP-C1 NCI-H292 cells extracts were obviously decreased,in transfecting pEGFP-C1-elafin NCI-H292 cells extracts.And MTT technique showed that compared with transfecting pEGFP-C1-elafin cells,adhesion capability of transfecting pEGFP-C1 cells with collagen were obviously decreased.Conclusion Transfection of recombinant human elafin gene into airway epithelial cells could maintain airway epithelium and enhance the capability of airway to inflammatory injury.

Objective To explore the mutual effect of proinflammatory factor tumor necrosis factor(TNF)-? and cigarette smoke extract(CSE) in the induction of mucin 5AC in BEAS-2B human airway epithelial cells.MethodsThe BEAS-2B cells were transfected with nuclear factor(NF)-?B decoy oligonucleotides(ODNs) or transfected with NF-?B scrambled ODNs as negative control,then treated with TNF-?,CSE,and TNF-? plus CSE,respectively. The expression of phosphorylated epidermal growth factor receptor(p-EGFR),mucin 5AC(MUC5AC) protein production and gene expression were detected by Western blot,ELISA and RT-PCR respectively.Results Significant increasing of p-EGFR protein production in scrambled ODNs transfected cells with elevation of MUC5AC protein production and mRNA expression was recorded and TNF-? and CSE co-incubated groups were much higher than single TNF-? or CSE stimulated group(P

Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P

Objective To synthesize a marine natural product, 24-methylenecholesterol. Methods The target molecule was synthesized in 7 steps using hyodeoxycholic acid as the starting material, 3?-(tetrahydropyran-2-yloxy)-cholest-5-ene-24-ketone as the key intermediate and isopropylation as the key step. Results The target molecule was synthesized with a total yield of 66% and confirmed by MS, IR, 1HNMR and 13CNMR. Conclusion Our synthesis of 24-methylenecholesterol is characterized by easily acquired starting material, short synthetic route, high yield and atom-economic.

Objective To study the mechanism of respiratory tracts inflammation induced by the particles with an aerodynamic diameter of less than 10 ?m (PM10) and to observe the effect of high exposed group. Methods Particles were collected at kitchen, smoking-room, roadside and lake-side (the control group). Suspension of PM10 and rat models treated with PM10, kitchen oil smoke, cigarette smoke, road dust were established with a control group. On 22th day, the counts of total leukocyte and neutrophils in bronchoalveolar lavage fluid (BALF) and superoxide dismutase (SOD), glutathion peroxidase (GSH-Px), malondiadehyde (MDA), Cytokin-induced neutrophil chemotactics (CINC) in lung homogenate were measured and histopathological examinations were conducted on lung tissues. Results The counts of total leukocyte, macrophage and neutrophils in PM10-treat groups and the count of eosinophilia in road dust group increased significantly than those in control group (P

Objective:To construct the highly efficient expression recombinant adenovirus that expresses elafin protein for further study.Methods:The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with pAdEasy-1 in BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and sequence scanned. Then the recombined adenovirus plasmid was digested with Pac Ⅰ and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein(GFP) expression. The expression of elafin protein was detected by Western blot and ELISA to appraise the function of elafin protein for oppressing the activity of elastic protease.Results:Digestion with restriction endonuclease, PCR, Western blot and ELISA confirmed that elafin gene was cloned into the adenovirus vector successfully.Conclusion:The recombined adenovirus Ad-elafin is constructed successfully, which will be benefit for future study.

Objective To determine the involvement of matrix metalloproteinase-9(MMP-9) in the signal transduction pathway of cigarette smoke-induced airway mucous hypersecretion. Methods The well cultured BEAS-2B airway epithelial cells were incubated with different concentrations of cigarette smoke extract (CSE),also some cells were preincubated with MMP-9 inhibitorⅠbefore CSE or exogenous epidermal growth factor(EGF) was added. The mucin(MUC)5AC mRNA level was analyzed by RT-PCR,MMP-9 and the phosphorylated EGFR(p-EGFR) protein production was assayed by western blot,MUC5AC protein and EGF protein were detected by enzyme-linked immunosorbent assay respectively,and gelatin zymography was used to determine the activity of MMP-9. Results Incubation of BEAS-2B cells with CSE up-regulated MUC5AC gene expression and protein production in a dose dependent manner (P0.05). Conclusion CSE stimulates the activity of MMP-9 and MMP-9 protein,causing the shedding of the EGF and leading to EGFR activation. This activation of EGFR downstream signaling pathway increases MUC5AC gene expression and mucin production.