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Objective:To develop an HPLC method for the simultaneous determination of vitamin B 1(thiamine), vitamin B2(ribo-flavin), vitamin B3(niacinamide), vitamin B5(pantothenic acid) and vitamin B6(pyridoxine) in complex vitamin B tablets.Meth-ods:An Alltima C18 column (250 mm ×4.6 mm, 5μm) was used for the separation , and 50 mmol· L-1 ammonium dihydrogen phos-phate ( adjusting pH to 3.0 with H3 PO4 ) and acetonitrile were used as the mobile phase with gradient elution at the flow rate of 0.5 ml · min-1 .The detection wavelength was set at 275 nm for vitamin B1 , vitamin B2 , vitamin B3 and vitamin B6 , and 210 nm for vitamin B5 .The column temperature was 30℃and the injection volume was 5μl.Results:The linear range of vitamin B 1 , B2 , B3 , B5 and B6 was 39.48-197.40, 40.16-200.80, 39.36-196.80, 38.80-194.00 and 41.76-208.80 μg · ml-1(r≥0.9999).The average recov-ery was 98.70％, 99.91％, 99.04％, 99.63％and 98.75％, and the RSDs were 0.40％, 0.27％, 0.40％, 0.92％ and 0.39％(n =9), respectively.Conclusion:The established method is accurate , simple and rapid, and can be utilized for the simultaneous determination of vitamin B 1 , B2 , B3 , B5 and B6 in complex vitamin B tablets .
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Objective To develope a high performance liquid chromatography ( HPLC) method for simultaneous determination of benzoic acid and salicylic acid in compound benzoic acid embrocation. Methods An Alltima C18 column (150 mm×4.6 mm,5 μm) was used for the separation, with methanol-0.1％ phosphoric acid (50:50) as the mobile phase at the flow rate of 1.0 mL?min-1 .The detection wavelength was set at 236 nm.The column temperature was 35 ℃ . Results The good linearity was obtained with the correlation coefficient (r) of 1.0000 for benzoic acid and salicylic acid;the precisions and stability were satisfactory, and the relative standard deviations (RSDs) were less than 0.3％.The average recoveries (n= 9) were 100.15％ and 99.85％ for benzoic acid and salicylic acid, respectively. Conclusion The established method is accurate, simple and rapid, and can be utilized for the simultaneous determination of benzoic acid and salicylic acid in compound benzoic acid embrocation.
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Teicoplanin is a kind of glycopeptide antibiotic extracted from actinomycetes after fermentation, which is mainly used for the treatment of multidrug-resistant gram-positive cocci infection. The domestic and overseas research literatures on the advances in the analytical methods for teicoplanin were consulted, reviewed and analyzed. The determination methods of teicoplanin mainly includ-ed in vivo and in vitro pharmaceutical analysis. The analytical methods involved high-performance liquid chromatography analysis, bio-assay determination, liquid chromatography-mass spectrometry analysis and micellar electrokinetic capillary chromatography, and a-mong them, HPLC technology was most commonly utilized in the determination of teicoplanin in biological samples and teicoplanin preparations. Much progress has been made in the analytical methods for teicoplanin, and further exploration of determination methods in vivo and in vitro will be beneficial to the quality control of the drug and guiding clinical rational administration.
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Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.
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Objective:Expression and purification of the α-HL of Staphylococcus aureus as antigen for making full human anti-α-HL antibody later ,providing of new treatment for Staphylococcus aureus infection.Methods:The total RNA of Staphylococcus aureus was extracted and the cDNA of α-HL was amplified by RT-PCR.The DNA of α-HL and pCold-TF plasmid was digested and ligated by T4 ligase and then transformed into E.coli TOPO 10.The recombinant plasmid α-HL/pCold-TF which verified by sequencing was trans-formed into E.coli BL21 for expression.The expression products was identified by SDS-PAGE and Western blot.Results: The size of amplified cDNA of α-HL was about 900 bp and the expressed soluble fusion protein of α-HL was about 90 kD(including the molecular chaperone in the vector ) after inducing expression for 24 h at 15℃.The Western blot results showed that the expressed protein was the fusion protein of α-HL.The purified α-HL was injected into BABL/c mice for making antiserum.The results showed that the antiserum had good binding activity with Staphylococcus aureus and the titer was greater than 10 000 times.Conclusion: The α-HL of Staphylococcus aureus was successfully cloned and the soluble fusion protein of α-HL was successfully expressed.
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Orthopedic robotics is an emerging industry in the area of healthcare , mainly used for minimally invasive treatment and accurate treatment .It can provide accurate surgical navigation and planning .Orthopedic robots can be mainly used for articular surgery , osteopathy surgery , spine surgery and traumatic orthopedics .This paper outlines the development and characteristics of orthopedic robots at home and abroad , analyzes the developments of orthopedic robots by combining medical imaging technology with clinical feedback , and predicts the future of this field .
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Objective To investigate the autophagy and apoptosis in acute myelogenous leukemia U937 cell induced by Sirolimus.Methods U937 cells were subcultured, and blank control group(normal) and Sirolimus treated groups(12 h, 24 h,48 h) were established.The Sirolimus treated groups were treated by 2 μmol/L concentration of Sirolimus for 12 h,24 h and 48 h, respectively.The cell morphology of U937 cells treated by Sirolimus was observed after 12 h,24 h and 48 h.The survival rate of cells was detected by cell counting kit-8 method.Cell apoptosis was detected by flow cytometry using Annexin V-FITC/PI double labeled.Real-time PCR was used to detect the level of mRNA expression in autophagy specific protein maker mictotubule-associated protein light chain 3 (LC3)-Ⅱ in different treated times by Sirolimus.Sirolimus LC3 protein expression levels after treatment were detected by Western blot method.Results Under inverted microscope, the cell number of Sirolimus treatment group reduced gradually after 12 h ,24 h and 48 h culture, volume of cells became smaller, cells got ruptured, and the nucleus pycnosis and cellular debris increased.With the extension of time, U937 cells survival rate was falling, and there was statistical differences compared with those of the control group(P =0.031).With Sirolimus treatment, U937 cells after 12 h,24 h and 48 h, U937 cell apoptosis rate increased, and there were statistically significances, compared with those of the control group (P =0.027).With Sirolimus treatment U937 cells after 12 h,24 h and 48 h,LC3-Ⅱ mRNA expression and protein expression were down-regulated compared with those of the control group, and there were statistically significances (P =0.029).Conclusions Sirolimus can induce autophagy and apoptosis in U937 cells.Autophagy protein LC3-Ⅱ in gene and protein expression levels were lowered, and LC3-Ⅱ may play an important role in regulating the leukemia cell autophagy.
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Objective:Expression of protein TSLP and selection of full human anti-TSLP single chain Fv ( scFv).Methods:The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results: The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion: The full human scFvs against for TSLP were selected suc-cessfully.
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Objective To investigate the expression of six-transmembrane epithelial antigen of the prostate (STEAP) in prostate cancer and its relationship with prostate specific antigen (PSA).Methods A retrospective analysis was performed of 65 consecutive patients verified for prostate canc-er by prostate biopsy or post-operational pathological examination.The clinical stage was classified ac-cording to TNM system and the pathological grade was classified according to Gleason score.STEAP expression in 65 prostate cancer samples (T1 9,T2 14,T3 17,T4 25;high differentiation 37,moder-ate differentiation 12 and low differentiation 16) was studied by using STEAP monoclonal antibody (Santa Cruz Biotech,USA) and SP immunohistochemical staining.Positive staining gray values were introduced to describe the intensity of STEAP expression.STEAP expression level was analyzed with respect to stage,grade,serum total PSA and free/total PSA (f/t PSA) ratio respectively.Results The serum total PSA concentration and f/t PSA ratio in all cases was (27.65±8.34) ng/ml and 0.15～0.04 respectively.STEAP was positively stained in 63 patients (T1 7,T2 14,T3 17,T4 25; high differentiation 37,moderate differentiation 11 and low differentiation 15).The mean STEAP-positive staining gray values in stage T1,T2,T3 and T4 of prostate cancer were 26.8%,45.6%,62.3% and 76.5%,respectively and in high,moderate and low differentiation group were 71.2%,52.8%,and 34.4% respectively.The positive staining gray values of STEAP expression was posi-tively correlated to the clinical stage of cancer and negatively correlated to the Gleason score and the ratio of f/t PSA (P<0.01,respectively).There was no significant relationship between STEAP ex-pression and serum total PSA concentration (P>0.05).Conclusion STEAP expression may act as one of new markers to the invasion degree and pathological grade of prostate cancer.
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4 ng/ml and/or with prostate nodule found on DRE or TRUS . Methods 197 patients suspected of prostate carcinoma underwent ultrasound guided sextant biopsy and 2～4 more cores in the nodule. Four patients whose initial biopsy was negative but subsequent PSA levels were continuously abnormal underwent repeated biopsies. Results Of the 107 cases with prostate nodules,in 34(31.8%) of them prostate cancer was detected,while in 90 cases without a prostate nodule, only 11(12.2%) were positive. Stratified analysis on PSA found that the higher the PSA, the higher the positive biopsy rate of prostate carcinoma. In the 4 cases with repeated biopsies, 1 prostate cancer was detected. Conclusions For patients with prostate nodule and/or PSA over 4 ng/ml,systematic biopsy should be taken, with 2 or more cores toward the nodule.In the patients with continuous high PSA levels,if the first biopsy is negative, rebiopsy should be taken.