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BACKGROUND:A steady method that can isolate endothelial progenitor cells(EPCs) need to be explored.Studies show that the EPCs riches in bone marrow with strong reproductive activity,which can be easily obtained.OBJECTIVE:To evaluate the isolating culture and biological characterization of EPCs derived from rat marrow.DESIGN,TIME AND SETTING:The in vitro cytology experiment was performed at the central laboratory of the First Affiliated Hospital of Chongqing Medical University from August to December 2008.MATERIALS:Twenty SD rats,with two-week-old,were obtained from Experimental Animal Center of Chongqing Medical University.METHODS:femurs and tibias of rats were isolated under sterile conditions,and then the bone marrow cavity was washed by 4 ℃ 0.01 mol/L phosphate buffer(PBS).Mononuclear cells were collected from marrow suspension by density gradient centrifugation,followed by cultured with M199 culture medium containing 20% fetal bovine serum to obtained cell suspension.The cells were vaccinated into culture flask(coated with fibronectin) with the density of 1?109/L,and incubated at 37 ℃ in 5% CO2 atmosphere.MAIN OUTCOME MEASURES:Morphologic change of cells was observed by an invert microscope,the expression of CD133,CD34 as well as KDR was detected by immunocytochemistry,and the flow cytometry was used to analyze the growth curve.RESULTS:The adherent cells formed clusters that were found to be characterized by density packed round cells surrounded by spindle-likes cells.Adherent cells were identified positive for CD133,CD34 and KDR by immunocytochemistry on days 4,7,10 and 14.CD133-positive cells reduced after 10 days.Flow cytometry analysis indicated that the number of CD133/KDR double positive cells gradually increased to the maximum at day 10 and then declined.KDR-positive cells gradually increased up with the time.EPCs growth curve showed that cells proliferated fast on days 3-6.CONCLUSION:EPCs can be isolated and cultured by density gradient centrifugation from marrow suspension of SD rats.These cells exhibit the characterization of endothelial cells,and can differentiate into endothelial cells.

Objective To search for the reasons of high positive rate of amotile bacteria and the diagnosis of septicemia in new-born Methods The blood was drawn from the different site of the new-born with septicemia and carricd out blood culture. The drug sensitivity test had been done by the method of paper stripdiffusion. The plasmids of bacteria were extracted rapidly by medified Birnboim method and the plasmid analyss was carried out. The plasmids's DNA of 35 epidemic strain was cut off by both restriction enzyme of Hind Ⅲ and EcoR Ⅰ. The outer membrane protein (OMP) was determined by SDS-polyacrylamide gel electrophoresis.Results There are 51 patients with positive blood culture amotile bacterium,of them, pollution; 35 cases (68．6%), septicemia: only 16 cases (31．4%),54．8% (57/104) strains bacteria have drug resistance to more of 12 drugs. 87．3% (165/189) strains bacteria have plasmids. They are cut off as 6 DNA fragments (1．9,2,4,5, 8．5 and 18Kb) by Hind Ⅲ restrietion enzyme. and as 5 DNA fragments (2,2．6,3．2, 6．3 and 22 Kb) by EcoR Ⅰrestrietion enzyme, it is showed that they come from a same clone. The epidemic strain include 10 slips OMP, but non-epidemic strain have 11 slip OMP, increase a 25Kd belt. The amotile bacteria with above-mentioned plasmid spectrum, restriction enzyme spectrum and OMP spectrum are only seen in the air, therapeutic dish and syringe needle.Conclusion The pollution is an important reason of amotile bactorium high positiye rate in new-born.Diagnosing septicemia should depend on bacteria culture, plasmid analysis restriction enzyme analysis of plasmid DNA, oMP determination and combining medical history and clinical manifestation.

Abstract: Objective　To screening new compounds that can inhibit the growth and biofilm formation of Staphylococcus aureus. Methods　Compounds that can inhibit the growth of Staphylococcus aureus were screened from the FDA approved drug library by 96 well plates. The absorbance value of 600 nm wavelength (OD600) was measured by Microplate Reader to detect the growth of Staphylococcus aureus planktonic cells in the culture supernatant. The minimum inhibitory concentration (MIC) of ozanimod against Staphylococcus aureus clinical isolates were detected by micro broth dilution method. The inhibitory effect of sub-inhibitory concentrations of ozanimod on the biofilm formation of Staphylococcus aureus was detected by crystal violet staining. Results　This study found that ozanimod could significantly inhibit the growth of Staphylococcus aureus SA113 (screening reference strain), and the MIC was 25.00 μmol/L. The MIC of ozanimod against 119 clinical isolates of Staphylococcus aureus [65 isolates of methicillin sensitive (MSSA) and 54 isolates of methicillin resistant (MRSA)] was 12.50 or 25.00 μmol/L. The MIC50 and MIC90 of ozanimod against the 119 Staphylococcus aureus isolates all were 25.00 μmol/L. This study found that 6.25, 12.50, 25.00 μmol/L of ozanimod could significantly inhibit the biofilm formation of 2 MSSA and 2 MRSA. The sub-MIC concentration of ozanimod (12.50 μmol/L) could significantly inhibit the biofilm formation of 14 MSSA and 11 MRSA, but had no inhibitory effect on the growth of planktonic cells of these Staphylococcus aureus isolates. Conclusion　Ozanimod can inhibit the growth of Staphylococcus aureus, including MRSA, and has good antibacterial activity. The sub-MIC concentration of ozanimod could significantly inhibit the biofilm formation of Staphylococcus aureus.

Objective To study on the effectiveness of endoscopic ultrasonography(EUS)in detec- ting insulinoma preoperatively.Methods Fifteen patients with clinical and biochemical signs of insulinoma were examined by EUS using a radial-scanning ultrasound endoscope and abdominal ultrasonography,CT, DSA prior to surgery.The outcome was evaluated on the basis of surgery and examination of the resected specimens.Results Fifteen patients with 16 lesions of insulinoma were identified by surgery and pathology. The aceuraey of diagnosis with EUS was 13/15(86.7%),and that with B-US,CT,DSA was 3/15(20%), 5/15(33.3%),9/14(64.3%)respectively.In the 14 lesions identified by EUS,10 lesions were depicted to be hypoechogenic,1 lesion was isoechogenic and 3 lesions were hyperechogenie.All 14 lesions were well demarcated and surrounded by normal pancreatic tissue.The minimum size of the lesion visualized by EUS was 0.5cm.Ten lesions were correctly detected by EUS with size of 0.5～2.0cm.EUS missed diagnosis in 2 lesions not for their small size.EUS falsely indicated a 10mm lesion from two lesions inside the head of pancreas.One lesion outside the pancreatic tail and one lesion in the pancreatic head were missed by EUS in another case.Conclusion EUS is superior in assessing the location of pancreatic insulinoma than other ima- ging methods such as B-US,CT,DSA.

The high-copy-number plasmid pcDNA3.1+ is unstable within S almonella typhimurium. A novel plasmid pmcDNA3.1+ was constructed by removin g the promoter sequence of ampicillin resistance gene (bla gene) in plasmid pcDNA3.1+. In contrast to pcDNA3.1+, pmcDNA3.1+ was stable within Salmonel la typhimurium SL7207 in LB medium with or without ampicillin. Further experi ments showed the ?-lactamase activity of Salmonella typhimurium SL7207(pmc DNA3.1+) was apparently lowered than that of Salmonella typhimurium SL7207( pcDNA3.1+) and the high ampicillin concentration was maintained longer in LB me dium culturing Salmonella typhimurium SL7207(pmcDNA3.1+). When mice were a dministered with Salmonella typhimurium SL7207(pmcDNA3.1+) intraperitoneall y, more than 95% of Salmonella cells separated from the spleen still harbore d the plasmid pmcDNA3.1+ 7 days later; but 99% of Salmonella cells lost the plasmid pcDNA3.1+ at day 3 in mice innoculated with Salmonella typhimurium SL7207(pcDNA3.1+). By lowering the expression of bla gene, the rapid deco mposition of ampicillin in LB medium was avoided and the metabolic pressure was relieved for the host cells. This method offers a solution for the problem of t he instability of high-copy-number plasmid within Salmonella typhimurium.

Objective To evaluate the value of cough test in the tension-free vaginal tape (TVT)procedure.Methods A cohort of 85 women with stress urinary incontinence underwent the TVT procedure with cough test (n =41) or without cough test (n =44).Patients in cough test group were performed according to the Ulmsten’s method strictly,with the stress of tape adjusted in light of cough test; whereas in other 44 operations,the tape was placed on the urethral tract without stress,and no cough test was performed.The urine catheter was removed after 48 hours postoperatively and follow-up evaluation was carried out at 12 month postoperatively.Results TVT procedure was carried out successfully in all patients by a single experienced surgeon.Four cases of urinary retention and 5 cases of voiding difficulty were observed in the cough test group.However,urinary retention or voiding difficulty was not detected in the nun-cough test group.Based on the twelve-month follow-up results,the cure rate was 92.6％ (38/41) in the cough test group and 93.1％ (41/44) in the non-cough test group.Flow-pressure study indicated that 11 cases in cough test group were in the obstruction zone,while only 3 cases in the obstruction zone were detected in the non-cough test group.Conclusions TVT is a safe as well as effective minimally invasive surgical procedure to treat female stress urinary incontinence.However,Adjusting stress of tape in accordance with cough test during the TVT may potentially increase the incidence of urinary dysfunction postoperatively.Therefore,no convincing evidence was gained to support the efficacy of cough test during TVT in terms of preventing postoperative voiding dysfunction.

Objective To explore the liver toxicities of the VDLD scheme in children with malignant tumor.Methods In a prospective trial,the levels of serum total protein,albumin, globulin,rate of albumin/globuin alanine aminotransferase,aspartate transaminase,gamma glutamyltranspeptidase,total bile acid and alkaline phosphatase were tested in children with malignant tumour before and after VDLD scheme,and compared with each other.Results The concentration of the serum total bile acid was significantly increased after VDLD scheme than before(P