Objective: To determine the content of tetrahydropalmatine in Yuanhuzhitong Dropping Pills by TLC scanning. Methods: The extraction solution of the sample was analyzed on silica gel G plate, with toluene anhydrous alcohol (20∶2, saturating for 30 min) as the developing system. The spot was located under UV (at 254nm), then detected by dual wavelength scanning (? S=280nm,? R=310nm). Results: The average recovery was 98.90%. RSD was 3.10%. Conclusion: The method is simple, accurate and available for the quality control of Yuanhuzhitong Dropping Pills.

Objective To evaluate and compare the histamine-releasing,potencies of cis-atracurium and atracurium during induction of general anesthesia.Methods Forty-five ASA Ⅰ or Ⅱ patients aged 16-71 yr undergoing elective surgery under general anesthesia were randomly divided into 3 groups (n=15 each):group Ⅰcis-atracurium (stored at 4-8℃)(group CIS1);groupⅡcis-atracurium (stored at room temperature)(group CIS2) and group Ⅲ atracurium (stored at 4-8℃)(group ATR).Anesthesia Was induced with TCI of propofol (Cp 3 μg/ml) and remifentanil (Ce 3-5 ng/ml).A bolus of cis-atracurium 0.15 mg/kg or atracurium 0.75 mg/kg Was given iv over 5-10 s as soon as the patients lost consciousness.Neuro-muscular block was monitored with TOF-Watch(R) SX(Organon,the Netherlands).Single stimulation (0.1 Hz) was apphed to the ulna nerve at wrist.The maximal degree of N-M block,onset time,duration of action and recovery index were recorded.The patients were intubated and mechanically ventilated when N-M block reached the maximal degree.The intubation condition Was evaluated.MAP and HR were continuously monitored.Changes in skin were scored (0=no change,Ⅰ=flushed>120 s,Ⅱ=erytbema,Ⅲ=urticaria).Blood samples were obtained before (T0,baseline),at 2 min after induction of anesthesia with TCI of propofol and remifentanil (T1) and 2 and 5 min after CIS/ATR administration (T2,T3) for determination of plasma histamine concentration using enzymatically amplified immunoassay.Results The onset time was significantly longer and the duration of action was significantly shorter in group CIS1 than in group ATR.The maximal degree of N-M block was 100%and the intubation condition was excellent in group CIS1 and ATR.There wag no significant difference in the recovery index between group CIS1 and ATR.The onset time was significantly longer and duration of action shorter in group CIS2 than in group CIS1.There was no significant difference in recovery index between group CIS1 and CIS2.There was no significant change in plasma histamine concentration at T1-3 as compared with the baseline at T0 in group CIS1 but plasma histamine concentration was significantly increased at T2,3 in group ATR.MAP was significantly decreased after induction of anesthesia with propofol and remifentanil,but CIS and ATR did not significantly change MAP.Conclusion The onset time is longer and duration of action is shorter after cis-atracurium than afar atracurium.The N-M block induced by cis-atracurium is significantly attenuated if stored at the room temperature.Cis-atracurium does not cause histamine release.

Objective To evaluate the effects of different storage periods at room temperature on domestic cisatracurium-induced neuromuscular (N-M) block.Methods One hundred and twenty ASA Ⅰ or Ⅱ patients,aged 18-64 yr,scheduled for elective operations under general anesthesia,were randomly divided into 3 groups (n =40 each):cisatracurium stored for 60 days at 4-8 ℃ group (group LT),cisatracurium stored for 30 days at room temperature group (group RT30) and cisatracurium stored for 60 days at room temperature group (group RT60).Anesthesia was induced with iv injection of midazolam and target-controlled infusion of propofol (target plasma concentration 3 μg/ml) and remifentanil (target effect-site concentration 3-5 ng/ml).A bolus of cisatracurium 0.2 mg/kg was given intravenously over 5-10 s as soon as the patients lost consciousness.N-M block was monitored with TOF-Watch SX (Organon,Netherlands).Single stimulation was applied to the ulnar nerve at wrist.The maximal degree of N-M block,onset time,clinical duration,recovery index and 75 ％ recovery time were recorded.The patients were intubated and mechanically ventilated when N-M block reached the maximal degree.The intubation condition was evaluated.Hypotension,bradycardia and skin allergy were recoded.Results Compared with group LT,no significant change was found in the onset time,clinical duration,recovery index and 75％ recovery time in group RT30 (P ＞ 0.05),and the onset time was significantly prolonged,clinical duration and 75％ recovery time were shortened in group RT60 (P ＜ 0.05).The onset time was significantly longer and clinical duration was shorter in group RT60 than in group RT30 (P ＜ 0.05).The intubation condition was excellent or good in the three groups and there was no significant difference among the three groups.There was no significant difference in the incidence of hypotension and bradycardia among the three groups (P ＞ 0.05).No patients developed skin allergy.The maximal degree of N-M block was 100％ in groups LT,RT30 and RT60 except one case with 95％ in group RT60.Conclusion No significant change is found in the N-M block induced by domestic cistracurium when stored for 30 days at room temperature,however,the N-M block is significantly attenuated when stored for 60 days at room temperature.

Objective To prepare stable aqueous dispersions of chitosan/multi-walled carbon nanotubes (CS/MWCNTs) composites,and observe the effects of CS/MWCNTs on the growth of human umbilical vein endothelial cells (HUVEC).Methods CS/MWCNTs composites were prepared by electrostatic interactions between negatively charged MWCNTs and positively charged low-molecular-weight CS.The prepared CS/MWCNTs were characterized by transmission electron microscopy and Zetasizer nano-analyser.The cellular uptake of the fluorescently labeled CS/MWCNTs was observed by laser confocal microscopy after incubating with HUVEC for 24 h at different concentrations.In vitro cytotoxicity and cellular reactive oxygen were also detected.Results When the mass ratio of low-molecular-weight CS to MWCNTs was equal or greater than 10∶1,the CS/MWCNTs can be stabilized in solution.Cellular uptake experiments showed that the CS/MWCNTs could enter into the cells and locate mainly in the cytoplasm.Cytotoxicity study showed that the CS/MWCNTs composites was less toxic than MWCNTs alone at high concentration (10 and 20 μg/ml).However,there was no significant differencein the level of cellular reactive oxygen between the two groups (P＜0.05).Conclusions CS/MWCNTs composites showed low cytotoxicity and high stability,which would be a promising carrier for drug delivery.

ObjectiveTo explore the endocytic pathway of TAT-LHRH modified chitosan/DNA nanoparticle (TLCDN) that exhibits high transfection efficiency and targeting to HepG2.MethodsPlasmid DNA was labeled with fluorescein,and the resulting fluorescent DNA was complexed with chitosan or TAT-LHRH modified chitosan to form chitosan/DNA nanoparticle (CDN) and TLCDN by the complex coacervation method.Internalization of TLCDN or CDN by HepG2 cells were measured in the presence of three kinds of inhibitors of endocytic pathway,Chlorpromazine,Filipin or Dynasore,using High-Content Analyzer to collect and analyze the data.ResultsChlorpromazine led to more decreased uptake of CDN than that of TLCDN,although not statistically significant.Filipin demonstrated significant inhibitory effect on the uptake of TLCDN while promoted the uptake of CDN.Dynasore resulted in a similar decrease in the uptake of both nanoprticles.ConclusionIt was demonstrated that CDN was taken up by HepG2 cells mainly through the clathrin-dependent endocytic pathway and TLCDN was more likely to be internalized by HepG2 cells through the caveolin-mediated endocytic pathway although the clathrin-dependent endocytic pathway was also involved.

Objective: To determine the contents of cinobufagin and resibufogenin in Qianglijiuxin Dripping Pills. Methods: The HPLC method was used at Kromasil C 18 column of 250mm?4.6mm, 5?m, 0.5% Dipotassium Hydrogen Phosphate Acetonitrile (50∶50) as the mobile phase, 1.0mL/min as the flow rate at detection wavelength of 296nm. Results: For cinobufagin, the linear range was 0.67?g～4.7?g and the average recovery rate was 97.78% with RSD=1.2%. For resibufogenin, the linear range was 0.33?g～2.3?g and the average recovery was rate 99.61% with RSD=2.0%.Conclusion: The method can be applied to content determination of cinobafagin and Resibufogenin in Qianglijiuxin Dripping pills.

Objective To investigate the change of serum osteoprotegerin level and relationships between serum osteoprotegerin level and coronary heart disease (CHD) in patients with type2 diabetes mellitus (DM) complicated with CHD.Methods One hundred and eight patients with type 2 DM complicated with CHD were selected as DM with CHD group,including 68 cases with acute coronary syndrome and 40 cases with stable angina pectoris.In addition,60 type 2 DM patients without CHD (DM without CHD group) and 40 healthy people (control group) were selected.Serum osteoprotegerin level was measured by enzyme linked immunosorbent assay.Results The serum osteoprotegerin level in DM with CHD group was significantly higher than that in DM without CHD group and control group [(4.12 ± 0.71)ng/L vs.(2.69 ± 0.52) and (2.14 ± 0.37) ng/L],and there were statistical differences (P＜ 0.05 or ＜ 0.01).In DM with CHD group,the serum osteoprotegerin level in acute coronary syndrome was significantly higher than that in stable angina pectoris [(4.56 ±0.92) ng/L vs.(3.61 ±0.76) ng/L],and there was statistical difference (P ＜ 0.05).The serum osteoprotegerin level in patients with Gensini score ＞ 40 scores (41 cases)was significantly higher than that in patients with Gensini score 20-40 scores (53 cases) and patients with Gensini score ＜ 20 scores (14 cases) [(4.92 ± 0.89) ng/L vs.(4.08 ± 0.75) and (3.39 ± 0.85) ng/L],and there were statistical differences (P ＜ 0.01 or ＜ 0.05).The serum osteoprotegerin level in patients with Gensini score 20-40 scores was significantly higher than that in patients with Gensini score ＜ 20 scores,and there was statistical difference (P ＜0.05).The serum osteoprotegerin level in single-vessel lesion (16 cases),double-vessel lesion (57 cases) and three-vessel lesion (35 cases) [(3.52 ± 0.82),(4.54 ± 0.68),(4.75 ±0.93) ng/L] were significantly higher than those in control group,and there were statistical differences (P ＜ 0.05); and the serum osteoprotegerin level in double-vessel lesion and three-vessel lesion were significantly higher than those in single-vessel lesion,and there were statistical differences (P ＜ 0.05) ;there was no statistical difference between three-vessel lesion and double-vessel lesion(P＞ 0.05).The results of Logistic regression analysis showed that glycosylated hemoglobin,total cholesterol,high sensitivity C reactive protein and osteoprotegerin were independent risk factors of type 2 DM complicated with CHD.Conclusions Serum osteoprotegerin level in patients with type 2 DM complicated with CHD is significantly increased.There is a significant association between serum osteoprotegerin level and the presence and severity of CHD,and the serum osteoprotegerin level is independent risk factors of type 2 DM complicated with CHD.

Objective To investigate the impact of chitosan and alkylated chitosan DNA nanoparticles on the function of human naive CD4+T cells.Methods The secretion of cytokines (IL-4 and TNF-γ) was observed after the co-incubation of human naive CD4+T cells with nanoparticles 12 h,24 h and 48 h,respectively.ResultsNone of the nanoparticles induced the production of cytokines ( IL-4 and TNF-γ ).Conclusion Chitosan and alkylated chitosan DNA nanoparticles will not induce the differentiation of human naive CD4+ T cells into T1 or T2 and may be considered as a safe gene carrier.

Objective To generate recombinant Pichia pastoris for high-copy expression of human tissue factor pathway inhibitor (TFPI). Methods The cDNA encoding human TFPI was inserted into the expression vector pPIC9K and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant plasmids were subsequently transformed into Pichia pastoris GS115 cells, and the transformants were confirmed by PCR amplification of the genomic DNA.The recombinant Pichia pastoris with high copies of TFPI cDNA was screened by G418 selection. Western blot and TFPI ELISA Kit were employed to analyzing. The temperature, time and concentration of methanol for the induction of recombinant protein were optimized. Results PCR analysis and DNA sequencing confirmed the successful construction of the expression vector rhTFPI-pPIC9K. Real time quantitative PCR and Western blot analysis demonstrated the positive correlation between TFPI expression level and the copy number of TFPI cDNA in Pichia pastoris cells. Optimization of the induction condition significantly elevated the expression level and activity of TFPI (9.95±0.78 mg/L and 3.91±1.37 U/mL). Conclusion The Pichia pastoris strain with high copy of TFPI expression was successfully constructed, which lays a solid foundation for the further investigation on the function of TFPI and its application in the prevention and therapy of diseases.

ObjectiveTo isolate and identify endothelial progenitor cells (EPCs) from human umbilical cord,and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro.MethodsEPCs were isolated from human umbilical cord in enzyme digestion method.The biological characteristics of EPCs were identified by flow cytometry and laser confocal microscope.The enhanced green fluorescent protein (EGFP) gene transfection mediated by EPCs was investigated using Lipofectamine 2000 as transfection reagent.ResultsEndothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later.These cellsdisplayed an improved positive expression of CD133 and kinase insert domain receptor (KDR).The endotheliallineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate (FITC)-UEA-1 binding and DiI-ac-LDL uptake assay with the aid of laser confocal microscope.The transfection results demonstrated high expression of EGFP taking EPCs as host cell.ConclusionEndothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions.EPCs demonstrated to be an ideal carrier for gene and cell therapy.