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

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 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 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 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 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 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