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OBJECTIVE:To establish a method for simultaneous determination of ibuprofen and indomethacin concentration in human urine. METHODS:The urine samples were precipitated by acetonitrile. HPLC method was adopted. The determination was performed on Discovery C18 column with mobile phase consisted of acetonitrile-20 mmol/L ammonium acetate solution(85:15,V/V, pH value adjusted to 3.5 with glacial acetic acid)at the flow rate of 1.0 mL/min. UV detection wavelength was set at 220 nm. The column temperature was room temperature,and sample size was 80 μL. RESULTS:The linear range of ibuprofen and indometha-cin were both 0.1-50.0μg/mL(r=0.9996,0.9995,n=3). The limits of quantitation were both 0.1μg/mL,and the limits of detec-tion were both 0.03 μg/mL. RSDs of inter-day and intra-day were all lower than 10％(n=5),and accuracy ranged 94.7％-97.2％. The extraction recoveries of ibuprofen and indomethacin were 89.5％-91.8％ and 90.2％-92.4％(all RSDs<10％,n=15),respec-tively. CONCLUSIONS:The method is simple and rapid with high selectivity,sensitivity and accuracy. It is suitable for simultane-ous determination of ibuprofen and indomethacin concentration in human urine.
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Objective To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2)in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. Methods Spontaneous abortion embryo tissues were collected,including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos(35 cases)from the Department of Gynaecology and Obstetrics of the Affilisted Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007.FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein;primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis.Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAIY2 genes in embryonic cells which have normal karyotypes;the groups were defined as the first experimental group(transfeeted with pshRNA-hsMAD2-1),the second experimental group(transfected with pshRNA-hsMAD2-2),the third experimental group(transfected with pshRNA-hsMAD2-3),the first control group(transfected with nothing),the second control group(transfected with pTZU6+1)and the independent group(transfected with pshRNA-N1).Interference efficiency was demonstrated by FQ-PCR and western blot:cell prolireration was meagured by methyl thiazolyl tetrazolium(MTT)assay;cell-cycle was assessed by flow cytometry (FCM):the chromosome numbers were calculated to analyze the variation of chromosomes.Results(1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue,twice or more spontaneous abortion tissue and indueed abortion tissue were 0.00879±0.00035.0.00901±0.00033 and 0.00941±0.00026 respectively,and there Wag no significant ditierence(P＞0.05)compared with each other;however,the protein levels of hsMAD2 in three groups were 0.2791±0.0311.0.0431±0.0020 and 0.5790±0.0331 respectively,and there were significant difierences(P＜0.05)compared with each other.(2)Recombinant shRNA plagmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic cells.Compared with the first control group(4%)and the second control group(3%),the recombinant shRNA could inhibit embryonic cell proliferation to 54% at 48 h after transfection(P＜0.05):compared with the first control group(8.2%)and the second control group(8.0%),the ratios of G2/M phase cells in the experimental group(17.9%)was significantly increased(P＜0.05);compared with the first control group (4.8%),the ratios of abnormal chromosomes in the experimental group was increased to 30.0%(P＜0.05).Conclusions Down-expression of hsMAD2 gene may be one of the mechanisms inducing numerical chromosome aberration,abnormal embryo development and the occurrence of spontaneous abortion.
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Objective To explore the mechanism of hBUB1 gene in the developing of spontaneous abortion embryos with numerical chromosomal abnormalitv.Methods Quantitative real-time RT-PCR and Western blot were used to determine the mRNA and protein level of hsMAD2 gene both in spontaneous abortion embryos with numerical chromosomal abnormality(experimental group)and with numerical chromosomal normality(control group).Recombinant shRNA plasmids targeting hBUB1 gene was constructed to inhibit the expression of endogenous hBUB1 genes in embryonic cells.Interference efficiency was demonstrated by fluorescent quantitative PCR and Western blot.The inhibitory rate of cell proliferation was measured by MTT assay and cells-cycle was assessed by flow cytometry.Resuns Western blot analysis showed that protein level of hBUB1 in the experimental group was decreased signifieandv(the rate of positivity and strong positivity were 8%and 93.5%,respectively,P＜0.05)compared with the control group.The expression of hBUB1 gene in embryonic cells was significantly and specially inhibited by shRNA plasmids (the mRNA level before and after treatment witll RNAi were 0.196±0.067 and 0.042±0.006,respectively,P＜0.05).The inhibitory rate of cell proliferation was increased to 62%from 4%at 48 h after transfection.The rate of G2/M phase cells was decreased after transfection with efficient shRNA(control group:40.2%and 41.3%,test group:21.3%).Conclusions Down-regulation of hBUB1 gene leads to the inhibition of cell proliferation and the arrest of cell-cycle.It also probably plays an important role in the development of spontaneous abortion embryos with numerical chromosomal abnormality.The clinical relevance warrant further investigation.
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OBJECTIVE:To establish a method for determination of the in vitro release rate of gatifloxacin carried by medical biofibrin glue (MBG-Gat).METHODS:The content of Gat was determined by HPLC on ZORBAX300SB-C18 column with column temperate kept at 30 ℃ and detection wavelength was 293 nm;the mobile phase consisted of potassium dihydrogen phosphate buffered solution (pH 3.5)-acetonitrile (76∶24) at a flow rate of 1 mL?min-1.The volume of injection was 20 ?L.MBG-Gat was placed in normal saline for drug release,with the release solution determined in every 12 h for consecutive 9 days.RESULTS:The linear range of Gat was 5～80 ?g?mL-1 (r=0.999 7) with an average recovery rate of 99.77%～103.53%,RSD≤2.77%(n=5).The accumulative release rate of MBG-Gat in normal saline within 9 days reach-ed above 70%.CONCLUSION:The established method for determination of the in vitro drug release of Gat is proved to be sensitive and reproducible,and it is applicable for the determination and analysis of Gat in vitro.The Gat carried by MBG is characterized by sustained release.
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Objective To construct the RNA interference eukaryotic expression vector targeting human centromere protein-I (CENP-I) and to observe its effect on the growth of human embryo kidney 293 cells (HEK 293). Methods The expression vectors of pGenesil-1/CENP-I-siRNA-1. pGenesil-1/CENP-I-siRNA-2 and pGenesil-1/CENP-I-siRNA-3 were constructed by gene recombination and then were transfected into the HEK293 cells by liposome. The expressions of CENP-I at the protein and mRNA levels were detected by Western blotting and fluorescence quality PCR (FQ-PCR). The effective vector and the best transfection time were selected. The growth and the cell cycle of the transfected cells were assessed by MTT assay and flow cytometry. Giemsa was used to stain the transfected cells to calculate the mitotic index. Results Sequence-specific siRNAs targeting CENP-I significantly down-regulated the expression of CENP-I in HEK293 cells. The recombinant plasmid of pGenesil-1/CENP-I-siRNA-3 was the effective vector. After transfecting for 72 h the best inhibited efficiency was achieved. In CENP-I-siRNA transfected cells,the rate of cell growth was decreased markedly. Cells at G 2/M phase and the mitotic index were increased conspicuous compared with the cells transfected with the blank vector or untransfected. Conclusion Down-regulation of CENP-I in HEK293 cells by sequence specific siRNA delays the cell growth and postpones the cell division.
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The pharmacokinetics of ranitidine was studied with the method of high performance li-quid chromatography in 10 healthy volunteers. The subjects were given a single dose of ranitidine 150mg (one capsule) orally. The serum concentration-time curves showed that one-compartment open model could be used in all subjects. The equation of mean serum conccentration was and with following pharmacokinetic parameters: (hr), and There were no statistically significant differences between the, kinetic parameters of reported articles and ours