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 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 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 mucin(MUC) expression induced by cigarette smoke.Methods The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by cigarette smoke extract(CSE),the cells were divided into 4 groups:negative control group,CSE treatment group,heroin pre-treatment group and ZnPPIX pre-treatment group.The expression of MUC5AC,epidermal growth factor receptor(EGFR),p-EGFR,HO-1 and dual oxidase 1 (Duoxl) were detected.The cell activity was assessed by methyl thiazolyl tetrazolium method.The change of HO-1 mRNA,Duoxl mRNA,EGFR mRNA and MUC5AC mRNA was examined by reverse transcriptase-polymerase chain reaction.The protein expression of EGFR,p-EGFR,Duox1 and HO-1 was measured by Western blot,while the pro tein expression changes of MUC 5 AC were detected by ELISA.Results The expression level of MUC 5 AC mRNA and its protein in the CSE group increased significantly (P＜0.05) as compared to those in the control group [0.412±0.043,(105±8) μg/mg.The mRNA and protein of EGFR,Duox1 and HO-1,the protein of p-EGFR increased significantly as compared to the control group.After the cells being pre-treated with hemin,the mRNA and protein of HO-1 increased significantly,while the mRNA and protein of Duox1,EGFR and MUC,SAC,the protein of p-EGFR decreased significantly as compared to the CSE group.After the cells were pre-treated with ZnPPIX,the increase of HO-1 mRNA and protein was not significant as compared to the control group,while the mRNA and protein of Duox1,EGFR and MUCSAC,the protein of p-EGFR increased significantly.Conclusion HO-1 decreased the level of Duox1,blocked ligand-dependent EGF-R activation and decreased the expression levels of MUCSAC.

Objective To explore the mutual effect of proinflammatory factor tumor necrosis factor (TNF)-α and cigarette smoke extract(CSE) in the induction of mucin 5AC in BEAS-2B human airway epithelial cells. Methods The BEAS-2B cells were transfected with nuclear factor(NF)-κB decoy oligonucleotides(ODNs) or transfected with NF-κB scrambled ODNs as negative control,then treated with TNF-α,CSE,and TNF-α plus CSE, respectively. The expression of phosphorylated epidermal growth factor receptor (p-EGFR) , mucin 5AC( MUC5AC) protein produc-tion and gene expression were detected by Western blot, ELISA and RT-PCR respectively. Results Significant in-creasing of p-EGFR protein production in scrambled ODNs transfected cells with elevation of MUC5AC protein production and mRNA expression was recorded and TNF-α and CSE co-incubated groups were much higher than single TNF-α or CSE stimulated group(P <0. 05). In NF-κB decoy ODNs transfected cells,TNF-α stimuli revealed an obvious reduction of MUC5AC protein production and gene expression. Conclusion TNF-α and CSE can syner-gistically induce mucin 5AC expression in BEAS-2B cells, transcription factor NF-κB plays a critical role in TNF-α induced mucin production but not as so important in mucin production by CSE stimulated cells.

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 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 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.