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Objective To investigate the bactericidal mechanism of electrolyzed oxidizing water (EOW) against Pseudomona aeruginosa (P.aeruginosa).Methods Bactericidal mechanism of EOW against P.aeruginosa was studied through intracellular protein leakage,nucleic acid,and cell membrane calcium ion permeability,2 ％ glutaraldehyde was used as positive control group,and normal saline (NS) was used as negative control group.Results The killing rates of EOW and 2％ glutaraldehyde to P.aeruginosa were both＞99.99％ with 30-second contact time,and 100.00％ with 60-second contact time.After 60-second contact with EOW,NS,and 2％ glutaraldehyde,the protein leakage of P.aeruginosa detected by bicinchoninic acid (BCA) were (96.00 ± 7.42),(94.15 ± 7.49),and (216.97 ± 10.35)μg/mL,respectively,difference was significant(F =613.20,P＜0.01),2％ glutaraldehyde group was higher than EOW group and NS group;protein leakage did not change with the increase of contact time(all P＞0.05).Electrophoretogram of random amplified polymorphic DNA showed high intensity dense band between 500-1000 Kb in EOW group and NS group,while 2％ glutaraldehyde group was without amplified bands.The fluorescence intensity of calcium ion of EOW group and 2％ glutaraldehyde group were both lower than that of NS group.Conclusion Bactericidal mechanism of EOW may be due to the damage of membrane permeability of P.aeruginosa,which causes Ca2+ leakage,but fails to cause protein leakage,the damage to nucleic acid is not obvious,DNA may not be a bactericidal target of EOW.
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Respiratory virus poses a serious threat to human life and health. Airway epithelial cells are the body's first line of defense from a wide variety of foreign pathogens, such as viruses and bacteria. Therefore, successful airway epithelial cell culture can provide a model for investigating the mechanisms underlying respiratory pathogenic diseases following airway virus infection. This respiratory disease model can also be used for the potential development of novel therapeutics. Here we provide a brief review of recent developments on the culture of cells derived from human trachea-bronchial airway epithelium, and the application of this model for studying respiratory virus and disease.
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Animals , Humans , Cell Culture Techniques , Epithelial Cells , Virology , Respiratory Tract Diseases , Virology , Virus Diseases , Virology , Virus Physiological Phenomena , Viruses , GeneticsABSTRACT
Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.
Subject(s)
Animals , Humans , Cell Differentiation , Dependovirus , Genetics , Epithelial Cells , Cell Biology , Metabolism , Green Fluorescent Proteins , Genetics , HEK293 Cells , Lung , Cell Biology , Membrane Potentials , Mucins , Metabolism , Swine , Transduction, Genetic , Tubulin , MetabolismABSTRACT
Objective To investigate the drug resistance status quo of imipenem-resistant Pseudomonas aeruginosa and carbap-enemase gene carrying in Xi′an area for guiding the clinical rational use of antibacterial drugs.Methods 151 strains of imipenem-re-sistant Pseudomonas aeruginosa isolated from clinical samples in 4 hospitals from August 2012 to July 2013 were continuously col-lected.Then the drug resistance characteristics of imipenem-resistant Pseudomonas aeruginosa were investigated by the antimicrobi-al drug sensitivity test.The PCR technique was adopted to detect the carrying situation of carbapenemase drug-resistance genes in imipenem-resistant Pseudomonas aeruginosa.Results Totally isolated 151 strains of imipenem-resistant Pseudomonas aeruginosa were mainly distributed in the neurosurgery ICU (37.1%),neurology ICU (27.1%)and the burn department (19.9%);the detec-ted strains were sensitive to polymyxin B and resistant to other 9 kinds of antibacterial drugs in different degrees;94 strains carried VIM gene,32 strains carried IMP gene,5 strains carried SPM gene and 3 strains carried SIM gene.Conclusion The multidrug re-sistance and pan-drug resistance phenomenon of imipenem-resistant Pseudomonas aeruginosa is serious,its cause might be related with the carbapenemases-producing drug resistant gene expression,the drug resistance genes are dominated by VIM and IMP.Clinic should strengthen the bacterial drug resistance monitoring and use antibacterial drugs rationally and effectively for preventing the spreading of imipenem-resistant Pseudomonas aeruginosa.
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Objective To investigate the distribution of characteristics,clinical manifestations,laboratory parameters and activity index of patients with systemic lupus erythematosus (SLE) and to determine their role in helping to make correct clinical diagnosis and disease the activity evaluation of SLE.Methods Collect the data of 1037 SLE patients of Ningxia Medical University Affiliated Hospital from January 2006 to June 2010.Data were analyzed with t test and Chi-square test.Results Over the past three years,there were more and more patients were admitted year by year.Among the 1037 cases of SLE patients,most of them 20-40 year-old woman,accounting for 67.5％ of the whole patient population,with a male to female ratio was 1:8.26.Joint pain was the most common initial symptom,accounting for 54.3％,followed by skin rash,accounting for 48.2％.Decreased complement C3 level and platelets counts, proteinuria,and positive anti-dsDNA antibody could be used as indicators for early diagnosis of SLE.SLEDAI activity score higher than 9 were presented in 26.0％ of patients.Factors that could impact the final score of SLEADI were fever,arthritis,skin rash,proteinuria,low complement levels,high titers of anti-dsDNA antibody,pleurisy,alopecia,mucosal ulcers,pericarditis,mental illness and decreased platelets count.Patients with active disease had a higher accidence of fever,arthritis,skin rash,lung damage,alopecia,mucosal ulcers,heart damage,mental illness and renal damaged,low complement levels,high level of anti-dsDNA antibody titers and elevated erythrocyte sedimentation rate.Conclusion SLE is a multi-system disease with multiple organ involvement,with characteristic clinical symptoms and immunological abnormalities,thus early diagnosis is very important.Understanding the characteristics of the diseases,correct judgement of the disease activity,reasonable and effective treatment all can delay the development of organ damage and improve the prognosis.
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Objective To establish animal models in order to provide an experimental study basis for both the pathogenesis study and taking effective prevention scheme for doxorubicin extravasation injury. Methods A total of 20 Kunming mice for experiments on doxorubicin extravasation injury were divided in-to four groups, I.e., high dose group(2 g/L), medium dose group(1 g/L), low dose group(0.5 g/L) and the control group (injection with water). Dosages were administered with subcutaneous injection on both sides of mice abdomen. The adverse reaction of body, damage areas of extravasation injury, recovery period were observed and histopathologic slides of animal models on both 4 days and 11 days after experiment were performed and compared. Results No significant adverse body reaction was observed after injection for all groups. The damage areas due to extravasation injury were dosage and concentration dependent. In addi-tion, significant differences in recovery period were observed for mice in different groups, that is, the higher injection concentration and dose led to the longer recovery period. Results from the histopathologic study in-dicated that the putrescence of damage area was developed in high dose group mice, and the ulcer occurred after 4 d of dosage in medium dose group mice, respectively. In contrast, no ulcer was observed in low dose group mice. Conclusions It would be feasible to establish a prevention model for mice on doxorubicin extravasation injury by subcutaneous injection at a dosage of 0.05ml(1 g/L).
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BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.