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 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 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

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 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 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 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.