OBJECTIVE:To establish a method for simultaneous determination of notoginsenoside R1,ginsenoside Rg1 and gin-senoside Rb1 in Shenqi xinshu capsule. METHODS:HPLC was performed on the column of Zorbax SB-C18(150 × 4.6 mm,5 μm) with mobile phase of acetonitrile-water at a flow rate of 1.0 ml/min,detection wavelength was 203 nm,column temperature was 30℃,and the injection volume was 10 μl. RESULTS:The linear range was 0.199 8-3.996 0 μg for notoginsenoside R1,0.842 8-10.143 0 μg for ginsenoside Rg1 and 0.823 4-9.978 0 μg for ginsenoside Rb1;RSDs of precision,stability and reproducibility tests were low-er than 2%;recoveries were 95.17%-100.17%(RSD=1.81%,n=9),97.32%-101.18%(RSD=1.44%,n=9)and 95.22%-98.89%(RSD=1.22%,n=9). CONCLUSIONS:The method is simple,accurate and reproducible,and can be used for the simultaneous contents determination of notoginsenoside R1,ginsenoside Rg1 and ginsenoside Rb1 in Shenqi xinshu capsule.

Objective In our previous work, the prevalence of anti-endothelial cell antibodies(AECA) in patients with systemic vasculitis and other autoimmune diseases was analyzed. AECA against a 47 000 endothelial cell antigen was found in patients of a variety of systemic vasculitis and systemic lupus erythematosus (SLE). It was suggested to be α-enolase by the combination of immunoblotting and proteomics methods. The aim of this work is to demonstrate that α-enolase is one of the targets of AECA, and to detect the prevalence of anti-α-enolase antibody in sera of patients with autoimmune disorders including systemic vasculitis. Methods The CDS of human Enol gene was amplified by polymerase chain reaction (PCR), with template of human placenta λzap express Cdna library. The product was then recombined with expression vector. After expression and purification from E.coli, the recombinant protein was analyzed by mass spee-trometry. The prevalence of anti-α-enolase antibody in patients with autoimmune disorders including systemic vasculitis was tested by Western blot and enzyme-linked immunosorbent assay (ELISA). Results The CDS of human Enol gene was subcloned to the expression vector. Recombinant human α-enolase was expressed and purified in E.coli. The recombinant protein was demonstrated to be his-tagged human a-enolase by mass spectrometry. Results of Dot-Blot revealed that the prevalence of anti-α-enolase antibody was 76.7% in systemic vasculitis [including 74.0% in Behcet's disease (BD), 81.5% in Takayasu artefitis (TA), 62.5% in Wegener's granulomatosus (WG), 92.3% in microscopic polyangitis (MPA) and 80.0% in Churg-Stranss syndrome (CSS)], 78.3% in SLE, 63.6% in Sjogren's syndrome (SS) and 78.9% in rheumatoid arthritis(RA). No positive signals were detected in sera of normal controls or patients with polymyositis/ dermatomyositis (PM/DM). There was no statistical significance among positive rates of anti-α-enolase antibody in systemic vasculitis, SLE, SS or RA patients. The prevalence of positive signals at the most extensive level (+++～++++) was 51.7% in patients with systemic vasculitis, 33.3% in SLE, 42.9% in SS and 20.0% in RA. There was statistical significant difference between RA and systemic vasculitis. Conclusion The identification of human α-enolase as one of the targets of AECA and its prevalence in a variety of autoimmune disorders will shed some light on the understanding of the pathogenesis of vascular injury in autoimmune diseases.

Objective To structure a preliminary indicators system for evaluating the projects of Hospital in finished phase.Methods With Delphi method,collect the experts' opinion of the importance of every item.With AHP (Analytic hierarchy process) method,calculate the weight coefficient of every item.Results The positivity of the experts was fine; The authority coefficient of two rounds were 0.859 and 0.833,the consultation results are reliable; The coordination coefficient of two rounds were 0.254 and 0.553,according to the significance test,p values were less than 0.05,indicating that the results were desirable.According to the score of every item,we got the weight index of every items based on the AHP method.Finally we structure the indicators system including 3 primary indicators and 10 secondary indicators.Conclusion The preliminary results of this study provide a reference for the performance evaluation of projects in finished phase of China-Japan Friendship Hospital.

Objective To prepare and screen out monoclonal antibodies against the receptor bind-ing domain (RBD) of Middle East respiratory syndrome coronavirus ( MERS-CoV) spike ( S) protein in mice. Methods The RBD of MERS-CoV S protein expressed in the insect-baculovirus system was purified and then used to immunize the female BALB/ c mice. The spleen cells collected from the mice were fused with myeloma Sp2 / 0 cells. The positive hybridoma cells were obtained by using limited dilution method. Enzyme-linked immunosorbent assay ( ELISA), Western blot assay and neutralization test based on the MERS-CoV pseudovirus were performed for further screening and identification. Results Twelve strains of hybridoma cells that produced the monoclonal antibodies against RBD of MERS-CoV S protein were screened out. All of the 12 monoclonal antibodies (McAbs) could have specific reaction with the RBD of MERS-CoV S protein as indicated by the results of ELISA. Of the 12 McAbs, two were identified as the immunoglobulin M (IgM) isotype and the rest were IgG1 isotype by using double antibodies sandwich ELISA. Four McAbs including 1F1, 2E4, 3C3 and 3E6 were identified as having neutralizing activity by the neutralization test based on MERS-CoV pseudovirus. Results of the Western blot assay showed that the four McAbs (1F1, 2E4, 3C3 and 3E6) could have specific reaction with the RBD of MERS-CoV S protein, but no cross-reac-tion with that of SARS-CoV S protein. Conclusion Twelve mouse-derived McAbs against the RBD of MERS-CoV S protein were obtained. The prepared hybridoma cells showed the characteristics of high speci-ficity and stability in antibody secretion. Four out of the 12 McAbs were proved to have neutralizing activity.

Objective To observe the effects of Shenmai injection on cardiac hemodynamics of isolated heart in rats of myocardial infarction model. Methods 30 myocardial infarction rat models created by ligation of left anterior descending coronary artery were randomly divided into a model group, a captopril group and a Shenmai group. 24 hours after successfully setup the models, all rat models were executed and their hearts were taken. Langendorff isolated heart perfusion method was adopted, and each group was perfused with 0.05mol/L amount of corresponding medicine, the model group was perfused with KH nutritive medium. left ventricular peak pressure(LVSP)and left ventricular ejection fraction(LVEF), left ventricular pressure increase/decrease rate(dp/dtmax±LV)(mmHg/s)and left ventricular end systolic diameter(LVESD)(%)and left ventricular end diastolic diameter(LVEDD)(%) was observed 5 min before the perfusion and 0.5 h, 1 h, 2 h after the perfusion. Results LVSP(9.47%± 0.15%vs. 3.34%± 0.05%),(11.25%± 1.31%vs. 3.79%± 0.04%) and LVEF reducing rate (7.44%± 0.10%vs. 4.94%± 0.04%), (10.24%± 0.31%vs. 5.34%± 0.05%) were elevated, and ±dp/dtmax(5 011 ± 253 mmHg/s vs. 5 827 ± 227 mmHg/s), (4 732 ± 212 mmHg/s vs. 5 837 ± 254 mmHg/s);(3 139 ± 127 mmHg/s vs. 4 722 ± 231 mmHg/s), (2 997 ± 125 mmHg/s vs. 4 793 ± 241 mmHg/s) was reduced in the Shenmai group 1 and 2 hours after the administration, which showed statistical difference compared to the model group (P<0.05). Conclusions Shenmai injection reduce myocardial contractility and intraventricular pressure of isolated heart o myocardial infarction rat models.

ObjectiveTo explore the application of the Structure Equation Model on validity assessment of the evaluation indicator system,we studied Medical Capital Development Fund projects in 2007 to evaluate their construct validity.MethodsPart of the evaluation results (1200 items) were divided into two parts randomly.PART1 was used for Exploratory Factor Analysis (EFA) to explore the Indicator System' structure,and formed model F1.Confirmatory Factor Analysis (CFA) was used to assess the construct validity of model F1.With considerations of Modification Index(MI) and reality,F1 was modified into F3.At last,we take PART2 as the sample to confirm F3.ResultThe results of model F3 fit indices showed a better fit.All the path coefficients,estimated load factors and other parameters were statistically significant.ConclusionStructural Equation Model is professional,reliable and satisfactory for the evaluation of indicator system's contract validity.

Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a novel human coronavirus and posed great threat to public health world wide,which calls for the development of effective and safe vaccine urgently. In the study, peptide epitopes tagrgeting spike antigen were predicted based on bioinformatics methods. Nine polypeptides with high scores were synthesized and linked to keyhole limpet hemocyanin (KLH). Female BALB/C mice were immunized with individual polypeptide-KLH, and the total IgG was detected by ELISA as well as the cellular mediated immunity (CMI) was analyzed using ELIs-pot assay. The results showed that an individual peptide of YVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI could induce the highest level of total IgG as well as CMI (high frequency of IFN-γ secretion) against MERS-CoV antigen in mice. Our study identified a promising peptide vaccine candidate against MERS-CoV and provided an experimental support for bioinformatics-based design of peptide vaccine.
Animals ; Antibodies, Viral ; immunology ; Computational Biology ; Coronavirus Infections ; immunology ; prevention & control ; virology ; Female ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Middle East Respiratory Syndrome Coronavirus ; genetics ; immunology ; Peptides ; administration & dosage ; genetics ; immunology ; Spike Glycoprotein, Coronavirus ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

To develop a recombinant lentivirus co-expressing structural protein of hepatitis C virus (HCV) and secreted Gaussia Luciferase (Gluc), we first constructed an expression vector that encoded HCV structural protein (C, E1, E2) and GLuc named pCSGluc2aCE1E2. The expression of HCV proteins and Gluc was confirmed by an immunofluorescence assay (IFA) and the detection of luciferase activity. Recombinant lentivirus (VSVpp-HCV) was developed by the co-transfection of pCSGluc2aCE1E2 into 293T cells with pHR'CMVA8.2 and pVSVG. The infectivity of VSVpp-HCV was confirmed by luciferase activity detection, IFA and western blotting. Virus-like particles were identified using electron microscopy after concentration. The results showed that the level of luciferase activity correlated with the expression of HCV protein after the infection of cells with lentivirus VSVpp-HCV. Therefore, the expression level of HCV proteins could be evaluated by detecting the luciferase activity of Gluc. In conclusion, this research pave a way for the development of transgenic mice that express HCV proteins and Gluc, which enable the evaluation of anti-HCV therapy and vaccine in vivo.
Animals ; Copepoda ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Hepacivirus ; genetics ; metabolism ; Hepatitis C ; virology ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; Cross Protection ; Female ; Genetic Vectors ; biosynthesis ; genetics ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Interferon-gamma ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; administration & dosage ; genetics ; immunology ; Viral Core Proteins ; administration & dosage ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; genetics ; immunology ; Viral Nonstructural Proteins ; administration & dosage ; genetics ; immunology

Objective To analyze the bone metabolism in hospitalized patients with Graves disease and the changes after 131I therapy.Methods The differences of bone metabolism were analyzed between 315 patients with Graves disease and 300 normal controls in a case-control study.The changes in bone turnover markers and BMD levels before and one year after 131I therapy were observed in 60 patients.Results Compared to normal control,bone turnover markers were markly higher and BMD levels were lower in patients with Graves disease.The level of thyroid hormones were positively related to bone turnover markers,while negatively related to total hip BMD (Z-score).But there was no linear relationship with lumbarand femoral neck BMD (Z-score).After one year of 131I therapy,bone turnover markers were markly lower than that before treatment,while BMD levels were partly higher than that before treatment.Conclusions In Graves disease patients,bone turnover markers were generally increased,while BMD levels decreased compared with normal people.After 131I therapy,along with the improvement of thyrotoxicosis,the high bone turnover rate can be suppressed,and BMD can partly recover.