Objective To investigate the effects of hyaluronan (HA) and CD44 on airway mucous hypersecretion,and explore the molecular mechanism of activation of epidermal growth factor(EGF)/epidermal growth factor receptor(EGFR) signal transduction pathway by signal factors. Methods BEAS-2 B airway epithelial cells were cultured in vitro,and were stimulated by neutrophil elastase(NE).Reactive oxygen species(ROS)scavenger(DMTU),hyaluronidase(Hase),CD44 antibody and tissue kallikrein(TK)inhibitor(PI)were served as interventional factors,and control group(serum free culture),NE stimulation group(50 nmol/L NE),DMTU+NE group(20μmol/L DMTU+50 nmol/L NE),DMTU+Hase+NE group(20 μmol/L DMTU+10μg/mL Hase+50 nmol/L NE),CD44Ab+NE group(5 μg/mL CD44Ab+50 nmol/LNE)and PI+NE group(1 00μg/mL PI+50 nmol/L NE)were established.The expression of mucin(MUC)5AC mRNA was detected by RT-PCR,the expression of MUC5AC and EGF protein was determined by ELISA,and the expression of phosphorylated epidermal growth factor receptor(p-EGFR)protein was analysed by Western blotting. Results The expression of MUC5AC,EGF and p-EGFR protein and MUC5AC mRNA in NE stimulation group was significantly higher than that in control group(P<0.01),the expression in DMTU+NE group was significantly lower than that in NE stimulation group(P<0.01),the expression in MTU+Hase+NE group was significantly higher than that in DMTU+NE group(P<0.05 for MUC5AC and p-EGFR protein and MUC5AC mRNA,and P<0.01 for EGF protein),the expression in CD44Ab+NE group and PI+NE group was significantly higher than that in NE stimulation group(P<0.05 for MUC5AC and p-EGFR protein and MUC5AC mRNA,and P<0.01 for EGF protein). Conclusion NE up-regulates the expression of MUC5AC gene via ROS/HA/CD44/TK/EGF/EGFR signal transduction pathway in airway epithelial cells.

Objective To clone the human mucin (MUC)5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. Methods The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software.After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced.By promoter deletion analysis, 3 promoter segments with diferent lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors.Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-кB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. Results Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end.The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully.Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-кB version (P＜0.05 vs. contro1) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. Conclusion This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression.There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.

Objective To investigate the effects of heme oxygenase-1 (HO-1) on the signal transduction pathway of cigarette smoke-induced mucin (MUC) expression in airway mucous hypersecretion. Methods The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by cigarette smoke extract (CSE),and treated with hemin or ZnPPIX,an inductor or inhibitor of HO-1. The expression of HO-1,dual oxidase 1(Duox1),MUC5AC,epidermal growth factor receptor (EGFR) and p-EGFR were detected. The cell viability treated with hemin or ZnPPIX was assessed by methyl thiazolyl tetrazolium method (MTT). The cells were divided into 4 groups:a negative control group,a CSE treatment group,an hemin pre-treatment group and a ZnPPIX pre-treatment group. The changes of EGFR mRNA,Duox1 mRNA,HO-1 mRNA and MUC5AC mRNA were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression changes of Duox1,HO-1,p-EGFR and EGFR were measured by Western blot,while the protein of MUC5AC in supernate and endochylema were detected by enzyme-linked immunosorbent assay (ELISA). Results The expression levels of MUC5AC mRNA and its protein in supernate liquid and endochylema in the CSE group were (0.660?0.044),(62.80?6.85) ?g/mg and (157.00?3.26) ?g/mg,and those of EGFR were 0.596?0.061 and 0.620?0.051,both of them increased significantly (P0.05),while the protein of p-EGFR,the mRNA and protein of EGFR,Duox1 and MUC5AC increased significantly (P

0.05).Conclusion Hypertonic stress can induce the airway epithelial cells to develop a high mucus secretion with the concentration-dependent manner in the level of transcription,P38 signal pathway plays a major role.

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.

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 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