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 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 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.

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 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 investigate the effect of protein inhibitor of activated signal transducer and activator of transcription 1 ( PIAS1 ) gene silencing on the inflammatory response of rat pancreatic acinar cell lines AR42J with cerulein stimulation, to study its role in the pathogenesis of acute pancreatitis.Methods The siRNA targeting PIASI was designed, synthesized, transfected into AR42J cells by lipofectmine 2000.24 h later, cerulean was added and cultured for another 24 h.Subsequent AR42J cells with cerulein stimulation were divided into 4 groups: cerulein, liposome, negative-siRNA and PIAS1-siRNA groups.In addition, a group with PBS was as control group.The activity of p38 mitogen- activated protein kinase (p38MAPK) was detected by western blotting.TNF-α, IL-1β, IL-6, matrix metalloproteinase (MMP) 9 expression were analyzed by RT-PCR and western blotting, respectively.Results The expression of p38MAPK in PIAS1-siRNA, negative-siRNA, liposome, cerulein,and control group was 1.93 ±0.11, 1.22 ±0.10, 1.30 ±0.17,1.32 ± 0.21, 0.12 ± 0.02;while the expression of phosphorylated p38MAPK was 2.10 ± 0.25, 1.36 ± 0.20,1.26 ±0.15, 1.23 ±0.25, 0.58 ±0.48, the expression in PIAS1-siRNA group was significantly increased when compared with other groups (P＜0.05).The levels of TNF-α, IL-1β, IL-6, MMP-9 mRNA were 1.66 ±0.15,1.66 ± 0.15,1.90 ±0.01, 1.56 ±0.20 in PIAS1-siRNA group, while the expression of protein was 2.06 ±0.37,2.20 ±0.34, 1.80 ±0.10, 1.17 ±0.05, which was markedly higher than those in other group (P ＜0.05).Conclusions PIAS1 gene silencing could enhance p38MAPK activity, and improve inflammatory mediator expression in pancreatic acinar cells with cerulein stimulation.

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.

Objective To evaluate clinical value of denaturing high performance liquid chromatography (DHPLC) used in detecting transforming growth factor beta receptor 3 (TGFBR-3) exons 11 and 12 polymorphism in women with idiopathic premature ovarian failure (POF).Methods From Feb.2009 to Dec.2011,110 patients with idiopathic POF undergoing treatment at Shenzhen Maternal & Child Health Institute affiliated to Southern Medical University were enrolled as POF group in this study.In the mean time,110 women under 40 years old with normal hormonal level and menstrual cycles as control group.The exons 11 and 12 of TGFBR-3 gene polymorphism were screened by using DHPLC,and results of DNA sequencing was as golden standard.Some related indexes were calculated,such as sensitivity,specificity,false negative value,false positive value,Youden index,positive predictive value,and negative predictive value.At the same time,20％ of the tested specimens were chosen randomly and detected by DHPLC again.The value of Kappa index were calculated by comparing the results between the first and second DHPLC analysis.Results The exon 11 of TGFBR-3 were not identified gene polymorphism and two nucleotide polymorphisms were identified in exon 12.For 2022 T/C polymorphism,the frequencies of CC with 0.9％ (1/110),TC with 22.7％ (25/110),TT with 76.4％ (84/110),Cwith12.3％ (27/220) and T with 87.7％ (193/220) in POF group were significantly different from CC with 0,TC with 9.1％ (10/110)and TT with 90.9％ (100/110),C with 4.5％ (10/220) and T with 95.5％ (210/220) in control group (all P ＜ 0.05).Allelic and genotypic frequencies of 2161-75 C/T were not differed significantly between the two groups (all P ＞ 0.05).As DNA sequencing as golden standard,DHPLC showed that the sensitivity was 100％,specificity was 97.9％,Youden index was 97.9％,positive predictive value was 96.3％,negative predictive value was 100％,and Kappa index was 0.888 (P ＜ 0.05).Conclusion DHPLC analysis is higher validity,reliability and practicability method in detecting TGFBR-3 polymorphism in idiopathic premature ovarian failure.

Objective To investigate the immunogenicity and cross protective effects of two novel HCV DNA vaccines in a mice model.Methods Two self-replicating alphavirus vector-based HCV DNA vaccines, pSCK CE1E2Y and pSCK H155, were constructed based on the genes encoding the structural pro-teins (Core, E1 and E2) and structural and NS3 fusion proteins (Core, E1 , E2 and NS3) of a HCV strain isolated from a Chinese patient (genotype 1b, Hebei strain), respectively.Western blot analysis was per-formed to detect the expression of fusion antigens.The BALB/c mice were intradermally immunized with the recombinant DNA vaccines by using electroporation.The immune responses induced in mice and the cross protective effects of the recombinant DNA vaccines were evaluated.Results The DNA vaccines effectively expressed the target antigens in vitro.The antigen-specific antibody responses and specific T cell immune re-sponses were induced in mice by the immunization of replicative DNA vaccines.However, no effective cross protection was provided by either of the DNA vaccines in the surrogate challenge model based on a recombi-nant heterologous HCV (JFH1, 2a) vaccinia virus strain.Conclusion Although no effective cross protec-tion was observed, both of the two replicative DNA vaccines could induce strong humoral and cellular im-mune responses against multi-target antigens of HCV strains.This study has paved the way for further inves-tigation on the development of novel HCV vaccines.