OBJECTIVE:To determinate the serum concentration of methotrexate by HPLC. METHODS:The samples were separated on a Waters C18 column at a column temperature of 30 ℃. The mobile phase consisted of acetonitrile-phosphate buffer(pH 7.2,12∶88) at a flow rate of 1.0 mL?min-1. The detection wavelength was set at 303 nm. RESULTS:The linear range of methotrexate was 0.5～80 ?g?mL-1(r=0.999 4). The methodological recovery rates were all above 90%; the intraday RSD was 3.03 %～3.52 % and the inter-day RSD was 2.57%～4.05%. CONCLUSION:The method is proved to be simple,rapid,accurate and sensitive,and it is suitable for the determination of serum concentration and pharmacokinetic study of methotrexate.

OBJECTIVE:To understand the distribution and drug resistance of the pathogens by sputum culture in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in our hospital so as to provide reference for rational use of antibiotics. METHODS:From Dec. 2010 to Dec. 2014,the sputum specimens were collected from the AECOPD patients,then the identification of 307 strains of pathogens and drug susceptibility test were carried out,and the data were analyzed statistically by using SPSS 17.0 software. RESULTS:A total of 307 strains of pathogens were collected,of which 17 cases of gram-positive ba-cillus accounted (5.54%),247 cases of gram negative cocci (80.46%),43 cases of fungi accounted (14.00%). The most com-mon isolates from sputum specimens were Pseudomonas aeruginosa (33.22%),Acinetobacter baumannii (19.54%),Stenotroph-omonas maltophilia(9.77%),Klebsiella pneumoniae(7.82%),Candida albicans(6.84%),etc. P. aeruginosa and A. baumannii were highly multidrug-resistant. There were 10 strains of ESBLs-producing K. pneumonia isolated, with the isolation rate of 41.67%. No Staphylococcus aureus strain was found resistant to vancomycin,teicoplanin or linezolid. Methicillin resistant strains in S. aureus(MRSA)accounted for 50.00%. CONCLUSIONS:Gram-negative bacilli are the most common pathogens in the AE-COPD patients. The common species of pathogens are highly resistant. More attention should be paid to the drug resistance monitor-ing of pathogens and rational use of antibiotics according to the results of susceptibility test.

Objective To investigate the effect of Toll-like receptor 2(TLR2)on the proliferation of human keratinocytes.Methods Keratinocytes were isolated from the foreskin of children,and subjected to primary culture.Atier 3-5 passages.the kemtinocytes were incubated with various concentrations of peptidoglycan(PGN).a TLR2 agonist.Cell proliferation was detected by MTT colorimetric assay and the suitable concentrations of PGN were determined.The mRNA and protein expressions of Ki67.TLR2.NF-kB p65 and TGF-α were detected by real-time quantitative PCR and Western blot.respectively,in keratinocytes treated witll PGN of 0,1.25,2.5 and 5 μg/mL.Antibody blocking test was utilized to evaluate the effect of blocking TLR2 with specific anti-TLR2 neutralizing monoclonal antibody before incubation with PGN on the expressions of Ki67,TLR2,NF-KB p65 and TGF-α by keratinocytes.Results The proliferation of kemtinocytes was significantly promoted by the incubation with PGN of 1.25,2.5 and 5μg/mL for 24 hours (all P＜0.05),which also increased the expression of Ki67 protein,TLR2 mRNA and protein,and NF-KB p65 protein.Further more,the mRNA expression of Ki67 in keratinocytes was elevated bv PGN of 1.25 and 2.5μg/mL,the mRNA expression of NF-KB p65 elevated by PGN of 1.25μg/mL,and the expressions of TGF-αprotein and mRNA elevated by PGN of 1.25 and 5μg/mL (P＜0.05).The mRNA and protein expressions of Ki67,TLR2,NF-kB p65 and TGF-αwere all inhibited by the blocking of TLR2 before incubation with PGN (a11 P＜0.05).Conclusion Activation of TLR2 bv PGN could induce the over-proliferation of human keratinocytes,likely through promoting NF-rB activation and TGF-α expression.

Objective To evaluate the method of controlled cortical impact(CCI) on long term cognitive disorder after traumatic brain injury(TBI) and to investigate the possible pathological mechanism.Methods Sixty male SD rats were randomly assigned into 3 groups:sham surgery group(n =10),control group (n =10) and CCI group(n =40).CCI application was used to make the bilateral frontal lobe controlled cortical impact model (depth:1.5 mm,velocity =3.5 m/s,dwell time =400 ms).Morris water maze test and Nissl's staining was used to assess the cognitive function and pathological changes after 8 weeks of CCI.The expressions of brain derived neurotrophic factor (BDNF) and tyrosine protein kinase B (TrkB) mRNA in frontal lobe and hippocampus tissue was detected by reverse transcription polymerase chain reaction(RT-PCR).Results The mortality in CCI group was only 12.5％.Morris water maze test results showed the escape latency in CCI group was longer than that in sham surgery and control groups(F =51.784,P ＜ 0.05).Percent of time spend in goal quarter during probe trial in CCI group was significantly less than that in sham surgery and control groups(F =13.468,P ＜ 0.05).Nissl's staining showed frontal lobe had obviously defects; Nissl's bodies of frontal cortex and CA1 region in hippocampus reduced.The expressions of BDNF and TrkB mRNA in frontal lobe and hippocampus were significantly less than those in sham surgery and control groups(P ＜ 0.05).Conclusions The CCI model can be applied for study on long term cognitive disorder after TBI with good stability and repeatability.Using the experimental parameters of CCI can damage the long term cognitive function after TBI in rats,and lead the pathology changes of brain tissue clearly.This may have some relationship with the expressions of BDNF and TrkB mRNA.

Objective To establish a rapid and precise continuous flow-injection chemiluminescence method for the determination of tetracycline and oxytetracycline. Methods In NaOH solution, tetracycline and oxytetracycline can sensitize obviously the chemiluminesence (CL) intensity of the reaction of luminol with KIO4, the sensitized CL intensity is proportional to the concentration of tetracycline and oxytetracycline. So, a new flow-injection CL method has been developed. The optimum chemical conditions for the CL reaction were investigated. Results Under the optimized conditions (KIO4 concentration: 1.0×10-5 mol/L; NaOH concentration: 0.1mol/L; luminol concentration: 1.0×10-4mol/L), tetracycline and oxytetracycline were determined. The linear range of the working curves was 1.0×10-7 -1.0×10-4g/mL, the detection limits was 1.0×10-8g/mL and 1.1×10-8g/mL, and the relative standard deviation was 2.6% (CS=1.0×10-6g/mL; n=11) and 2.0% (CS=1.0×10-6g/mL; n=11) respectively. Conclusion The method is simple, rapid, and sensitive, and it has been successfully applied to the the determination of tetracycline and oxytetracycline tablets, the mean recoveries being 99.7% and 98.8% respectively.

Objective To establish a rapid and precise continuous flow-injection chemiluminescence (CL) method for the determination of perphenazine. Methods In HNO_3 medium, perphenazine could be oxidated by ceriuim (IV) and CL was proportional to the perphenazine concentration without any sensitizers. Thus, a new flow-injection CL method was developed. Results Under the optimized conditions, the proposed method allowed the determination range within 1.0?10~ -7 -7.0?10~ -5 g/mL with the detection limit of 8.0?10~ -8 g/mL. Eleven parallel assays were conducted on perphenazine of 1.0?10~ -6 g/mL, with the relative standard deviation of 1.8%. Conclusion The method is simple, rapid, precise, and sensitive, and has broad linear range; therefore, it has been applied to the the determination of the perphenazine in tablets with satisfactory results.

Objective To determine if preventive antibiotics is effective in stroke associated pneumonia in patients with acute stroke.Methods Medline (January 1950 to January 2017),EMBASE (January 1974 to January 2017),Cochrane Library (January 2009 to January 2017),CNKI (January 1979 to January 2017)and Wanfang data (January 1998 to January 2017) were searched for randomized controlled trial comparing preventive antibiotics with placebo/blank controls for stroke associated pneumonia in patients with acute stroke.The included studies were screened out strictly based on the criterion of inclusion and exclusion.The quality of included studies was evaluated and the data were extracted by two researchers independently.RevMan 5.1 was used for Meta analysis.Results A total of 4 studies involving 3894 patients were included.The results of Meta-analysis indicated that there was no significant difference in the incidence of stroke associated pneumonia between preventive antibiotics and control groups (OR=0.96,95％CI:0.72-1.29,P=0.810);and there were no statistically significant differences in mortality (OR=1.05,95％CI:0.88-1.25,P=0.570) and good outcome (modified Rankin scale ≤ 2,OR=1.02,95％CI:0.89-1.17,P=0.780).There were no serious adverse reactions related to the studied drugs in 4 studies.Conclusion Preventive antibiotics could neither reduce the incidence of stroke associated pneumonia nor decrease the mortality or improve the proportion of good outcome.