Objective To supply scientific basis for the reasonable development and utilization of Rubus multibracteatus Levl.et Vant.resources.Method The total flavonoids was extracted from Rubus multibracteatus Levl.et Vant.with 60% methanol and its content was determined by spectrophotometry.The reliability and repeatability of the method were tested through stability,repeatability,accuracy and recovery experiments.Result Both standard and tested solutions had the maximum absorbance at 510 nm.The RSD values of repeatability,accuracy and recovery experiments were 2.11%,0.97% and 2.4% respectively.The results of content determination showed that the total flavonoids in Rubus multibracteatus Levl.et Vant.was 3.8%.Conclusion Extracted with 60% methanol,colored with aluminiumnitrate and determined by spectrophotometry at 510 nm were suitable for the determination of total flavonoids in Rubus multibracteatus Levl.et Vant..

AIM　To investigate the effects of ginkgolide B on arachidonic acid (AA) metabolizing enzymes and the level of intracellular calcium in rat polymorphonuclear leukocytes. METHODS　Intracellular free calcium was quantitated by Fura-2 fluoresence technique. Phospholipase A2 (PLA2) activity was determined by incorporating 3H-arachidonic acid in leukocytes. 5-Lipoxygenase (5-LO) activity was evaluated by RP-HPLC. RESULTS　In comparison with control, ginkgolide B at final concentration of 0.1-10 μmol*L-1 inhibited A23187 induced AA release by 10.9%-22.2%; at final concentration of 0.1-50 μmol*L-1, ginkgolide B inhibited LTB4 and 5-HETE synthesis stimulated by PAF by 29.4%-88.8% and 26.2%-89.3% respectively. At the final concentration of 0.1-100 μmol*L-1, ginkgolide B decreased the rise of intracelluar calcium level induced by pletelet activating factor (PAF) and N-formyl-methionine-leucine-phenglalanine (fMLP) by 13.9%-51.4% and 2.2%-36.6%, respectively. CONCLUSION　Ginkgolide B was found to significantly inhibit PLA2 and 5-LO activities, as well as the increase of the intracellular calcium induced by PAF.

Objective Mongolian gerbil can make themself urine concentration for saving water and adapt to the harsh desert environment, due to their very unique moisture control system in the body.Methods Mongolian gerbil is resistant to drought on account of their special kidney. Histology of the kidney, ureter and bladder in Meriones Unguiculataus were observed by light microscopy using HE staining.Results The results showed that compared with rats and mice, the Mongolian gerbils have more developed distal tubules, and well developed inner renal medulla.Conclusions We hope that the findings of this study enrich our understanding of the histology of urinary system in Mongolian gerbils and provide support for the laboratory animalization of this animal.

Objective To investigate the effects of remifentanil on sinoatrial (SA) node autorhythmicity in rabbits.Methods Twenty-four healthy rabbits of both sexes weighing 1.8-2.2 kg were sacrificed.Their hearts were removed and sinoatrial nodes were dissected and placed in Tyrode solution saturated with 95 ％ O2-5 ％ CO2 at 36 ℃.The action potentials of the sinus node pacemaker cells were recorded by intracellular glass microelectrode technique.The experiment was performed in 3 parts (n =8 each).Part Ⅰ:the sinoatrial node was exposed to remifentanil 2,4,8,16 and 32 ng/ml respectively.The action potentials were recorded after the sinoatrial nodes were exposed to each concentration of remifentanil for 15 min.Part Ⅱ and Ⅲ:the sinoatrial nodes were first exposed to Ca2+ channel agonist Bay K8644 0.5 μmol/L or K+ channel blocker TEA 20 nmol/L for 15 min.Then remifentanil was added until the concentration reached 16 ng/ml (final concentration) and 15 min later the action potentials were recorded.The action potential parameters included,amplitude of action potential (APA),rate of pacemaker firing (RPF),action potential duration at 90％ repolarization (APD90) and velocity of diastolic depolarization (VDD).Results Remifentanil significantly decreased,APA,RPF,VDD and prolonged APD90 in a concentration dependent manner as compared with the baseline values.Pretreatment with Bay K8644 could block the effects of remifentanil on SA node pacemaker cells,while TEA did not affect the electrophysiologic effects of remifentanil on SA node pacemaker cells.Conclusion Remifentanil exerts a negative chronotropic action on SA node pacemaker cells.These effects are likely produced by decrease in Ca2+ current,while opening of K + channels is not involved in these effects.

Objective To investigate the effects of remifentanil on lung ischemia-reperfusion (I/R) injury and expression of angiotensin-converting enzyme (ACE) mRNA in rats.Methods Forty pathogen-free adult male Sprague-Dawley rats,weighing 300-350 g,were randomly divided into 5 groups (n =8 each):sham operation group (group S),group I/R and different doses of remifentanil groups (groups R1-R3).Lung I/R was produced by occlusion of the left hilum of lung for 45 min followed by 120 min reperfusion.Remifentanil was infused intravenously at a rate of 0.2,0.6 and 1.2 μg·kg-1 ·min-1 until 120 min of reperfusion after a loading dose of 1 μg/kg at 15 min before occlusion of the left hilum of lung in groups R1,R2 and R3,respectively.The equal volume of normal saline was given in groups S and I/R.The rats were scarified at 120 min of reperfusion and lungs were removed for microscopic examination and determination of wet/dry lung weight ratio (W/D ratio) and the expression of ACE mRNA (by RT-PCR) in the lung tissue.The pathological changes of the lung were scored.Results Compared with group S,the pathological score and W/D ratio were significantly increased and the expression of ACE mRNA was up-regulated in groups I/R,R1 and R2,and the pathological score was significantly increased (P ＜ 0.05),and no significant change was found in W/D ratio and the expression of ACE mRNA in group R3 (P ＞ 0.05).Compared with group I/R,the pathological score and W/D ratio were significantly decreased and the expression of ACE mRNA was down-regulated in groups R2 and R3,and the pathological score and W/D ratio were significantly decreased (P ＜ 0.05) and no significant change was found in the expression of ACE mRNA in group R1 (P ＞0.05).Compared with group R1,the pathological score and W/D ratio were significantly decreased and the expression of ACE mRNA was down-regulated in group R3,and the pathological score and W/D ratio were significantly decreased (P ＜ 0.05) and no significant change was found in the expression of ACE mRNA in group R2 (P ＞ 0.05).The pathological score,W/D ratio and expression of ACE mRNA were significantly lower in groups R3 than in group R2 (P ＜ 0.05).The pathological changes of the lung were significantly reduced in groups R1-R3 as compared with group I/R.Conclusion Remifentanil can attenuate lung I/R injury in rats in a dose-dependent manner and down-regulation of ACE mRNA expression may be involved in the mechanism.

Objective To compare the sensitivity, specificity and consistency of five kits for quantitative detection of hepatitis B virus (HBV) DNA. Methods Four domestic fluorescence kits, A, B, C, and D, and Roche Cobas Ampliprep/Cobas TaqMan HBV test for quantitation of HBV DNA in serum, were applied to detect the National Standards, an additional plasma for sensitivity (7 times dilution), 15 plasma samples from healthy blood donor and 45 plasma samples from chronic hepatitis B patients. Results All five kits showed the correct results for the 9 positive specimens and 8 negative specimens from the National Standards. The quantitative results of specimens for sensitivity met requirements of the National Standards. The lowest concentration of HBV DNA by these three kits was 15.6 IU/mL. The lowest detection level of HBV DNA for domestic kit B was ≤500 IU/mL. There was linear correlation between the results by Roche kit and domestic kit C (r> 0.98). Kit D showed 2 false-negatives results in the 15 healthy blood donor samples. The coincidence rates between 4 domestic kits and the Roche kit ranged from 50% to 96% (A: 61%, B: 50%, C: 96%, D: 83 %). The consistency rate of kit C with the Roche kit was significantly higher than those of kit D, A and B (X2=5.62, P<0.05, X2=28. 93, P<0.01, X2=44.31,P<0.01, respectively). The consistency rates among these 5 kits were highest when testing samples with HBV DNA levels between 1×104-1×107 IU/mL. Conclusions The qualities of domestic kits vary remarkably and kit C has the greatest consistency rate with the Roche kit. Therefore, the qualities of domestic kits need to be further improved.

Objective To investigate the correlation of the expression of high mobility group protein B1 (HMGB1) and blood-borne metastasis in patients with pancreatic carcinoma. Methods The serum HMGB1 levels were determined by Western blot analysis in 68 patients with pancreatic cancer, 18 patients with chronic pancreatitis and 21 healthy controls. Among the 68 patients with pancreatic cancer, the serum HMGB1 levels of 37 patients before and after operation were compared. The expression of HMGB1 and CD31 were detected by immunohistochemistry in 67 patients with pancreatic carcinoma. Results The serum HMGB1 levels in patients with pancreatic cancer was (119.7±54.5) ng/ml, which was significantly higher than (40.2±25.5) ng/ml in chronic pancreatitis (P<0.001) and (13.1±4.3) ng/ml in healthy control (P< 0.001). The serum HMGB1 levels in patients with pancreatic cancer before operation was (120.2±8.2) ng/ml, which was significantly higher than (69.3±5.1) ng/ml after operation (P <0.001). The expression rate of HMGB1 in pancreatic cancer tissues was 43.6%. The expression of HMGB1 were significantly related with tumor differentiation, TNM stage, blood-borne metastasis, and vascular density(P <0.01). The HMGB1 positive tumor cells were adjacent to the blood vessels with lumen, and the rate of HMGB1 expression in intravascular tumor cells was 71%. Conclusions The HMGB1 was highly expressed in pancreatic cancer tissues. HMGB1 expression positive pancreatic carcinoma cells were prone to invade blood vessels with lumen which may be related to blood-borne metastasis in pancreatic cancer.

BACKGROUND:Mesenchymal stem cells (MSCs) exist in human tissues.Presently,cell source is single;culture method has great differences;obtained results are not consistent.Thus,it cannot verfy that isolated and cultured cells are identical calls,which is difficult to compare.OBJECTIVE:To compare the biological features of MSCs derived form bone marrow (BM),perpheral blood (PB) and cord blood (CB) under in vitro culture conditions.DESIGN,TIME AND SETTING:The cytological in vitro controlled study was performed at the Department of Hematology,Navy General Hospital of Chinese PLA from June 2007 to December 2008.MATERIALS:A total of 10 donors of hemopoietic stem cell transplantation at the Department of Hematology,Navy General Hospital of Chinese PLA were selected.MB and PB cells were obtained from the same donor,and cell volumes were respectively 20 mL and 2 mL.CB cells (30 mL) were obtained from healthy primipara at the Department of Obstetrics,Navy General Hospital of Chinese PLA.METHODS:MSCs were obtained from BM,PB and CB by Percoll density gradient + adherence method,and then incubated in DMEM/F12 medium containing 10% fetal bovine serum.When 80%-90% confluency,cells were digested in trypsin-EDTA and made into 5×10~8/L cell suspension as P_0.Above-described operation was performed as P_1,and the rest may be deduced by analogy as P_2-P_5.MAIN OUTCOME MEASURES:The following parameters were measured:cell growth morphology;results of Wright-Giemsa staining;results of cytochemistry;cell proliferation amount;cell surface markers using flow cytometry.RESULTS:Time of adherence,time to 50% confluency and time to 80% confluency of BMSCs were earlier comarped with the PBMSCs and UCMSCs.Adherent cells from BM grew in whirpool-like type,while CB and PB did not at 5-7 days.Majority of aderent cells from BM were fibroblast-like cells,and small parts were endothelioid cells.Aderent cells from PB and CB at the fifth generation contained more endothelioid cells and mononuclear and macrophage-like cells besides fibroblast-like cells.PAS stain,Sudan black B stein,alkaline phosphatase (AKP) staining of adherent cells from BM,PB and CB were negative from P_1 to P_5.Compared with P0 cells,number of BMMSCs till P5 was significantly more in PBMSCs and UCMSCs (P ＜ 0.05).Positive rates of CD29,CD44,CD90,CD71,CD105,CD166 and HLA-ABC were 55.9% 92.8% at P0 to P5,but ≤6% following BMMSCs were incubated;19.7%-33.4% at P0 to P5,but ≤10% following PBMSCs were incubated;35.4%-93.2% at P_0 to P_5,but ≤20% following CBMSCs were incubated.Positive rates of CD34,CD45 and HLA-DR were low in BM-,PB-and CB-MSCs.Positive rates of CD14 and CD31 were low in BMMSCs;12.1%-28.3% in PBMSCs,and 8.1%-21.3% in CBMSCs.CONCLUSION:MSCs can be attained from BM,PB and CB.Quantities of MSCs form BM are the highest,with single component,followed by CBMSCs and PBMSCs,with multiple components.

Objective To assess the incidence of thrombocytopenia and abnormal coagulation,door to needle time (DNT),and safety in patients with ischemic stroke who receive intravenous thrombolytic treatment prior to the availability of blood platelet (PLT) and coagulation results.Methods Consecutive acute ischemic stroke (AIS) or transient ischemic attack patients within 12 hours of symptom onset who were admitted from January 2009 to March 2013 were retrospectively recruited.First laboratory reports in their medical charts were collected to assess the incidence of thrombocytopenia and abnormal coagulation.In the mean time,consecutive AIS patients who received intravenous thrombolysis at Huashan Hospital during the same period were retrospectively recruited.The thrombolytic procedures were further optimized since June 10.3760/cma.j.issn.1006-7876.2014.07.00626,2012.With informed consent,intravenous thrombolysis was initiated without PLT and coagulation results after certain previous history and medications were ruled out.Thrombolytic patients were divided into two groups based on the initiation of thrombolysis before or after PLT and coagulation results.Baseline demographic data,symptomatic intracerebral hemorrhage rates,mortality on the 7th day as well as functional outcome at 3 months were collected.DNT as well as efficacy and safety of thrombolysis therapy were compared between the two groups.Results Of 298 AIS patients within 12 hours of onset,8 had thrombocytopenia or abnormal coagulation.One hundred and twenty cases of intravenous thrombolysis patients were recruited.Waiting for PLT and coagulation results prolonged DNT than without waiting for them (90 min vs 59 min; U =870.000,P ＜0.01).There was no statistically significant difference in the rate of symptomatic intracerebral hemorrhage (sICH),early efficacy,7 d mortality and 3-month good outcome between two groups.Conclusions The incidence of thrombocytopenia and abnormal coagulation is low in AIS patients.Initiating intravenous thrombolysis prior to the availability of coagulation results can shorten DNT,while increased risks of sICH and 7 d mortality were not observed.

Objective To determine factors associated with favorable clinical outcome in acute ischemic stroke patients who received intravenous thrombolysis.Methods Patients treated with intravenous recombinant tissue plasminogen activator (rt-PA) between January 1,2008 and May 31,2013 were recruited.Favorable outcome was defined as modified Rankin scale (mRS) score ＜ 2 at 3 months.The baseline characteristics were compared by univariate and multivariate analysis.Results Of all the 148 patients studied,within the 4.5-hour time window,the rate of patients with a favorable outcome was 52.4％ (65/124),significantly better than those beyond the time window(20.8％ (5/24),x2 =8.048,P =0.005).Univariate analysis showed that age (U =2 146.000,P =0.025),TOAST classification (x2 =11.412,P =0.010),Oxfordshire Community Stroke Project (OCSP) classification (x2 =17.409,P =0.001),baseline blood glucose (U =1 446.500,P ＜ 0.01),baseline fibrinogen (U =1 689.000,P =0.038),admission NIHSS score (U =1 140.000,P ＜ 0.01),24-hour NIHSS score (U =458.000,P ＜0.01),and early symptom improvement (24-hour NIHSS score decreased ≥ 4 points,or down to 0 point;x2 =19.576,P ＜ 0.01) were the factors associated with clinical outcomes.Multivariate analysis revealed that age (odds ratio (OR) =0.943,95％ confidence interval (CI) 0.892-0.996,P =0.024),baseline fibrinogen (OR =0.448,95％ CI 0.208-0.963,P =0.040),baseline NIHSS score (OR =0.749,95％ CI 0.663-0.845,P ＜ 0.01),and early symptom improvement (OR =14.970,95％ CI 4.460-50.249,P ＜ 0.01) were independent predictors of clinical outcomes.Conclusions The effect of intravenous thrombolysis on clinical outcomes of acute ischemic stroke patients is time-dependent.Age,baseline NIHSS score,baseline fibrinogen level,and early symptom improvement are independent predictors of clinical outcomes after intravenous thrombolysis.