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 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 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 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 This single-center prospective,case-controlled study was carried out to investigate clinical effects on patients with aging face who underwent facial autologous fat grafting with platelet-rich plasma (PRP).Methods Thirty patients with facial sagging or partial depression and requiring autologous fat grafting were randomly selected for the study.Photograph and 3D scanning were taken before and 3-month after the operation.Patient and physician satisfaction was rated using Visual Analogue Scale (VAS) ranging from 1 (most unsatisfactory) to 10 (most satisfactory)3 months after the operation.Statistical difference between the patient and physician satisfactory scores was analyzed.Results The average physician VAS score was 7.9 ± 1.0 while the average patient VAS score was 8.0±1.2.The scores between two groups had no significant difference (P＞ 0.05).Four patients developed bruising and swelling postoperatively that disappeared less than 7 days later.There was no feed back of serious complications from patients.Further investigation of patients with scores less than 7 showed the main reason of low patient satisfaction was that the improvement of facial depression did not meet the requirements of the patient,and fat absorption led to unsatisfactory filling.Conclusions The high satisfactory scores indicate good aesthetic result of facial autologous fat grafting with PRP.PRP may lead to higer survival rate of fat and satisfaction.

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 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 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.