Objective To design a set of feasible program of data monitor based on the database's security of the "No.1 Military Medical Project" in many years of running successful operation to enhance safety audit of database.Methods The system was consisted with date's collection of active and passive for safety information,preservation of backup data,and analysis of credibility dictionaries and auditing.Results The complete processing program of network security monitor which could form alarm message and advance corresponding disposal plans was came into being and its better reliability.Conclusion By using the way,the question of the "No.1 Military Medical Project" in database monitoring is solved in network environment and ensured safe operation of database,the network security of the "No.1 Military Medical Project" system is promoted.

To establish a LC-MS/MS method to determine the concentrations of liquiritin, glycyrrhizin, glycyrrhetinic acid, amygdalin, amygdalin prunasin, ephedrine, pseudoephedrine and methylephedrine of Maxing Shigan decoction in rat plasma, and study the differences on their pharmacokinetic process in normal rats and RSV pneumonia model rats. After normal rats and RSV pneumonia model rats were orally administered with Maxing Shigan decoction, the blood was collected from retinal vein plexus of different time points. Specifically, tetrahydropalmatine was taken as internal standard for determining ephedrine, while chloramphenicol was taken as internal standard for determining other components. After plasma samples were pre-treated as the above, the supernatant was dried with nitrogen blowing concentrator and then redissolved with methylalcohol. The chromatography was eluted with mobile phase consisted of acetonitrile and 0.1% formic acid solution in a gradient manner. ESI sources were adopted to scan ingredients in ephedra in a positive ion scanning mode and other ingredientsin a negative ion scanning mode. The multiple-reaction monitoring (MRM) method was developed the plasma concentration of each active component. The pharmacokinetic parameters of each group were calculated by using Win-Nonlin 4.1 software and put into the statistical analysis. The result showed the plasma concentration of the eight active ingredients, i.e., liquiritin, glycyrrhizin, glycyrrhetinic acid, amygdalin, amygdalin prunasin, ephedrine, pseudoephedrine and methylephedrine within the ranges of 1.04-1040, 1.04-1040, 0.89-445, 1.05-4200, 1.25-2490, 0.3-480, 0.3-480, 0.3-480 microg x L(-1), with a good linearity and satisfactory precision, recovery and stability in the above ingredients. After modeling, except for glycyrrhetinic acid whose pharmacokinetic parameters were lacked due to the data missing, all of the rest components showed significant higher Cmax, AUC(0-1) and lower clearance rate (CL) than that of the normal group, indicating the increase in absorption in rats in the pathological state by reducing the clearance rate. The method is accurate and sensitive and so can be used to determine the plasma concentrations of the eight active ingredients in Maxing Shigan decoction. RSV pneumonia-infected rats absorbed more ingredients in Maxing Shigan decoction.
Animals ; Chromatography, High Pressure Liquid ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacokinetics ; Male ; Pneumonia, Viral ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Respiratory Syncytial Virus Infections ; drug therapy ; metabolism ; Tandem Mass Spectrometry

OBJECTIVETo observe the effect of Bushen Huoxue Recipe (BHR) on inhibiting vascular calcification (VC) in chronic renal failure (CRF) rats by regulating BMP-2/Runx2/Osterix signal pathway, and to explore its possible mechanism.

METHODSThirty SD rats were randomly divided into the normal group, the model group, and the BHR group, 10 in each group. Rats in the model group and the BHR group were administered with 250 mg/kg adenine suspension by gastroagavage and fed with 1.8% high phosphorus forage, once per day in the first 4 weeks, and then gastric administration of adenine suspension was changed to once per two days in the following 5-8 weeks. Rats in the BHR group were administered with BHR at the daily dose of 55 g/kg by gastrogavage in the first 8 weeks, once per day. Equal volume of normal saline was given to rats in the normal group by gastrogavage for 8 weeks. Histological changes in renal tissue and aorta VC were observed by HE staining and alizarin red staining respectively. Levels of calcium (Ca), phosphorus (P), serum creatinine (Cr), blood urea nitrogen (BUN), and intact parathyroid hormone (iPTH) in serum were detected. Protein expression levels of bone morphogenetic protein (BMP-2), Runt related transcription factor (Runx2) , and Osterix were detected by Western blot.

RESULTSHE staining showed that compared with the normal group, disordered glomerular structure, tubular ectasia and dropsy, intracavitary inflammatory cell infiltration, dark brown crystal deposition in kidney tubules, renal interstitial fibrosis, and decreased number of renal blood vessels in the model group. Compared with the model group, normal glomerular numbers increased more, reduced degree of tubular ectasia, decreased number of inflammatory cells, and reduced adenine crystal deposition in the BHR group. Alizarin red staining showed that compared with the normal group, calcified nodes could be found in the model group, with extensive deposition of red particle in aorta. Compared with the model group, calcified nodes were reduced in the BHR group. Compared with normal group, serum levels of P, SCr, BUN, and iPTH significantly increased, serum Ca level significantly decreased, protein expressions of BMP-2, Runx2, Osterix also increased in the model group (P < 0.05, P < 0.01). Compared with the model group, serum levels of P, SCr, BUN, and iPTH levels significantly decreased, serum Ca level significantly increased, protein expressions of BMP-2, Runx2, Osterix also decreased in the BHD group (P < 0.05, P < 0.01).

CONCLUSIONBHD could improve renal function, Ca-P metabolism, and renal histological changes in CHF rats, down-regulate the expression level of BMP-2/Runx2/Osterix signal pathway in vascular calcification of CRF, which might be one of the mechanisms for inhibiting VC in CHF.

Animals ; Blood Urea Nitrogen ; Bone Morphogenetic Protein 2 ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; pathology ; Kidney Failure, Chronic ; drug therapy ; metabolism ; Kidney Function Tests ; Kidney Tubules ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcription Factors ; metabolism ; Vascular Calcification ; drug therapy

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.
Antibodies, Monoclonal, Murine-Derived ; pharmacology ; Antibody-Dependent Cell Cytotoxicity ; Antigens, CD20 ; immunology ; Genes, Reporter ; Humans ; Reproducibility of Results ; Rituximab

AIM:To observe the effects of sevoflurane preconditioning on brain injury in hypoxic mice and its possible mechanism. METHODS:Male C57BL/6J mice were randomly divided into control (C) group, hypoxia (H) group,2% sevoflurane preconditioning for 30 min + hypoxia(S1+H) group,2% sevoflurane preconditioning for 60 min+hypoxia (S2+H) group and 4% sevoflurane preconditioning for 30 min + hypoxia(S3+H) group. The hypoxia model was established by continuous inhalation of(6.5±0.1)% O2for 24 h. The sevoflurane preconditioning treatments,S1,S2 and S3,were conducted by inhalation of 2% sevoflurane for 30 min,2% sevoflurane for 60 min and 4% sevoflurane for 30 min,respectively,with the carrier of(21.0±0.5)% O2,followed by washout for 15 min and then hypoxia treatment. The histological changes of the hippocampal CA1 area were observed under light microscope and transmission electron micro-scope(TEM),and serum lactate dehydrogenase (LDH) activity was measured by colorimetric method. Furthermore, the protein levels of erythropoietin (EPO) and vascular endothelial growth factor(VEGF) in brain tissue homogenate were ex-amined by ELISA,and the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) and gluta-thione peroxidase(GPx) were measured by microplate reader. RESULTS:After hypoxia for 24 h,cell edema or pyknosis in the hippocampal CA1 area was observed in H group. Sevoflurane preconditioning reduced hypoxic injury, and the cell ultrastructure under TEM was significantly improved in S2+H group. Compared with C group,the serum LDH activity and the levels of EPO,VEGF and MDA in brain tissues were significantly increased in H group,while the activity of SOD and GPx decreased. After sevoflurane pretreatment,the serum LDH activity and the levels of EPO and VEGF in brain tissues were lower than those in H group,and the most significant difference was observed in S2+H group. Moreover, the MDA content and SOD activity decreased,and the GPx activity increased in the sevoflurane preconditioning groups. CONCLU-SION:Sevoflurane preconditioning attenuates brain injury in hypoxic mice by regulating antihypoxic protein synthesis and reducing oxidative stress.

Objective To investigate the correlation between the CT perfusion and microvessel density (MVD),expression of vascular endothelial growth factor(VEGF)in neoplasm of head and neck.Methods Eighty-eight lesions of head and neck were scanned by spiral CT.The largest axial surface of the mass was searched on unenhanced imaging,and at this level the dynamic contrast enhanced scan series was acquired.Time-density curves (TDC)were created from circular or oval regions of the interest drawn over the mass,target artery by Toshiba Xpress/SX spiral CT with perfusion functional software.The parameters were measured including:peak height (PH ),peak time (PT ),mean transit time (MTT), contrast enhancement ratio(RPH),and perfusion flow (PF).Histopathological slides of 35 masses were carefully prepared for the anti-CD34 and VEGF immunohistochemical staining and tumor microvessel density and calculation of VEGF expression scores.The parameters of CT perfusion were correlatively study with MVD and VEGF.Results(1)The TDC of CT perfusion imaging could be classified into 3 types.The TDC of 53/77 (68.9% )malignant tumors presented the type with rapid ascending and rapid descending after injecting contrast.The TDC of 6/9 malignant lymphomas showed low platform curve。(2)The PF median of thyroid carcinoma was 82.2(41.0,183.4)ml?min~(-1)?100 g~(-1).There was significantly difference in the parameters of CT perfusion among thyroid carcinoma and squamaous cell cancer (Median 23.8 (7.0, 108.4)ml?min~(-1)?100 g~(-1))and lymphomas (Median 24.5(13.2,78.6)ml?min~(-1)?100 g~(-1)).(3) MVD in benign tumors was (44.7?3.4),and in malignant tumors,it is (49.6?14.8 ).There was no significantly difference in MVD between benign and malignant tumors.High VEGF expression was found in 15 malignant tumors and 1 benign tumors,low VEGF expression was found in 9 malignant tumors and 10 benign tumors.(4)There were no significantly difference in VEGF expression and MVD.There was good correlation between MVD (M 40.0 )and PH (M 26.9 ),RPH (M 14.5 ),PF (M 46.8 )(r = 0.35,45.49, 0.41 ).There was correlation between VEGF(M 4.0)and MTT(M 16.7 )(r = -0.41 ).Conclusion The TDC and CT perfusion could be helpful to differentiate benign from malignant tumors. CT peffusion in neoplasm of head and neck is correlated with MVD and VEGF,and may reflect MVD and expression of VEGF.

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.
Antibodies ; analysis ; chemistry ; Gas Chromatography-Mass Spectrometry ; methods ; Glycosylation ; Immunoconjugates ; analysis ; chemistry ; Maleimides ; analysis ; chemistry ; Molecular Weight ; Pharmaceutical Preparations ; analysis ; chemistry ; Spectrophotometry, Ultraviolet ; methods

OBJECTIVETo investigate the mechanisms of angiotonin II (AngII)-induced Ca(2+)-independent pathways mediated by Rho kinase in hepatic stellate cells (HSCs).

METHODSHSC-T6 cells were treated with 1 micromol/L of AngII, and the subsequent cell contraction was directly observed with silicone rubber membrane culture method. The cells with 10 micromol/L AngII treatment were examined for myosin light chain (MLC) phosphorylation level using Western blotting, and the effects of irbesartan (a specific inhibitor of AngII 1- receptor) and Y27632 (a Rho kinase inhibitor) on AngII-induced MLC phosphorylation were evaluated. RT-PCR was used to detect the expression of Rock2 in Ca(2+)- independent pathways mediated by Rho kinase.

RESULTSAngII induced HSC contraction and time-dependent MLC phosphorylation changes, which peaked 15 min after the treatment followed by gradual reduction. Irbesartan or Y27632 treatment significantly lowered MLC phosphorylation level in AngII-induced cells (P<0.01). The mRNA expression of Rock2 increased significantly after AngII treatment (P<0.01), but decreased following subsequent irbesartan or Y27632 treatment.

CONCLUSIONAngII induces HSC contraction through Ca(2+)-independent pathways mediated by Rho kinase.

Angiotensin II ; pharmacology ; Animals ; Blotting, Western ; Cell Movement ; physiology ; Cell Shape ; drug effects ; physiology ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Phosphorylation ; drug effects ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; rho-Associated Kinases ; biosynthesis ; genetics ; metabolism

Objective To investigate the effect of miR-218-1-3p on the proliferation,cycle,and apoptosis of A549 cells in non-small-cell lung cancer.Methods miR-218-1-3p was transfected into non-small cell lung cancer A549 cells by LipofectamineTM 2000 Reagent,and the expression of miR-218-3p was detected by real-time PC R.Invasion and migration were assayed using the Transwell method.The effect of miR-218-1-3p on the proliferation of A549 cells was assayed by the MTS method.Changes in the cell cycle and apoptosis of A549 cells transfected with miR-218-1-3p was detected by flow cytometry.Changes in indicators related to cell proliferation,cycle,and apoptosis were detected by fluorescence quantitative PCR.Results Compared to the control group,the cell proliferation of A549 cells was significantly inhibited (P ＜ 0.05) and the proportion of cells in the S and G2-M phases was significantly decreased when miR-218-1-3p was up-regulated.In addition,compared with the control group,the early apoptotic rate was significantly increased by up-regulating miR-218-1-3p.We further detected indicators related to cell proliferation,cycle,and apoptosis and found that CYCLIN-D1 and BCL-2 were significantly downregulated.Conclusion miR-218-1-3p may inhibit proliferation,induce cell cycle arrest,and promote cell apoptosis of non-small cell lung cancer A549 cells by regulating CYCLIN-D 1 and BCL-2.

The macroporous microspheres were prepared through suspension polymerization and based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate.The effect of porogen on the microspheres structure was evaluated in terms of pore size and surface area.Porogen contained dichloromethane (δ=9.7 (cal/cm3)1/2) and N-octanol (δ=10.3 (cal/cm3)1/2) which corresponded to a good and poor solvent,respectively.The solubility parameter of porogen was controlled in the range of 9.89-10.09 (cal/cm3)1/2.The pore size of microspheres increased with the difference value of solubility parameter between the polymer and the porogen.On the contrary,the surface area of microspheres decreased in this study.The anion exchange media was prepared through coupling poly(ethylene imine) in the microspheres,and the proteins transport was determined by frontal analysis method.The macroporous microspheres with 257 nm pore size could still afford a high proteins capacity (45.1 mg/mL).These macroporous supports showed a large potential in a rapid separation of proteins.