AIM: To optimize the extraction of the anthraquinone aglycnes from Rhubarb using supercritical fluid extraction(SFE). METHODS: Orthogonal design and variance analysis were used to optimize five operational variables of SFE. RESULTS: The optimized operational variables of SFE were obtained as following: the temperature was 70 ?C , the pressure was 35 MPa, the static extraction time was 8 min, the dynamic extraction volume 5 mL and methanol were used as modifier. CONCLUSION: SFE is rapid, convenient and accurate, and can be used to extract athraquinone aglycones from Rhubarb.

Objective:To explore the effectiveness and safety of levosimendan treating severe decompensated heart failure (SDHF) .Methods :A total of 80 patients with SDHF caused by various caused were selected ,randomly and equally divided into levosimendan group and dobutamine group .Both groups received intravenous drip of corre‐sponding drug based on routine treatment ,and the course was 24h .After two weeks ,therapeutic effects were com‐pared between two groups .Results:After two weeks ,total effective rate of levosimendan group was significantly higher than that of dobutamine group (95.0% vs .72.5% , P= 0.005) .Compared with dobutamine group ,after two weeks , there were significant reductions in dyspnea status score [ (2.57 ± 0.80 ) scores vs . (1.89 ± 0.70 ) scores] ,general status score [(3.23 ± 0.65) scores vs .(2.95 ± 1.29) scores] ,respiratory frequency [(24.32 ± 0.98) times/min vs .(20.6 ± 1.58) times/min] and level of brain natriuretic peptide [(1584 ± 325.63) mg/ml vs .(1011.92 ± 302.31) mg/ml] ,and significant rise in left ventricular ejection fraction [(40.16 ± 4.85)% vs .(46.53 ± 3.37)% ] in levosimendan group , P<0.05 or <0.01. Conclusion:In short-term therapeutic effect on severe decompensated heart failure ,levosimendan is superior to dobutamine ,it possesses good safety and tolerance ,which can be used as an effective drug for severe decompensated heart failure .

Objective To learn the present pain management quality at tertiary hospitals in China and the application of the quality evaluation system for acute pain management quality recommended by American Pain Society.Methods Analyzing the present pain management at five tertiary hospitals in China by using the evaluation system recommended by American Pain Society,questionnaires and medical records reading.Results The hospitals were found with setbacks in describing pains,recording pains with tools,using the right pain controls,using non-drug pain control measures and pain management outcomes; differences were found between the acute pain service group and non-acute pain service group in pain management quality,as the pain impacts of both groups on activities,emotion and sleep were 3.12±2.82 and 5.16±2.07 (P＜0.05) respectively.Conclusion Setbacks exist in both the process and outcomes of post-surgery pain management at these hospitals; those with acute pain service management are better than those without in terms of post-surgery pain management quality.The evaluation system recommended by American Pain Society is scientific,sensitive,practical and operable,extensively applicable to evaluation of acute pain management quality at hospitals in China.

Objective To construct an eukaryotic expression plasmid PeDNA3.1 (+)/Lpp20 and to detect its expression in HeLa cells, and to observe the humoral and cellular immune responses in C57BL/6 mice induced by the Helicobacter pylori Lpp20 DNA vaccine injected intramuscularly. Methods The Lpp20 gene was amplified by PCR. PCR product was subcloned into the eukaryotic expression vector pcDNA3.1 (+)/ Lpp20, and the recombinant plasmid was transfected into HeLa cells using Liposome. After verifying that the Lpp20 antigen gene could be expressed in HeLa cells. Six weeks old C57BL/6 mice were immunized with pcDNA3.1 (+)/Lpp20 or pcDNA3.1 (+) or PBS buffer intramuscularly at 2-week interval for four times. ELISA was used for the quantitative detection of the specific IgG antibody in the sera of C57BL/6 mice and the cytokine IFN-γ in mice spleen lymphocyte culture medium after stimulating by Lpp20. The proliferation response of spleen cells was detected by MTT assay. The Lpp20 gene in muscle was identified by PCR. Results The significant specific antibody titers were detected by ELISA in DNA vaccine groups and the highest titer was 1:1024 after 6 weeks. The cytnkine IFN-γ in mice inoculated with pcDNA3.1 (+)/Lpp20 was increased and reached (410.36±56.23) pg/ml. A significant difference was tested between the experiment group and the control group[(25.26±10.85)pg/ml] ,P <0.01. The proliferation response of spleen cells of DNA vaccine group(SI: 2.37±0.22) was significantly higher than those of mice injected with pcDNA3.1 (+) (SI:1.53+0.47) ,P<0.01. Lpp20 gene could exist constantly in musculature cells of mice. Conclusion The eukaryotic expression recombinant pcDNA3.1 (+)/Lpp20 was successfully constructed. Strong humoral and cellular im-munity can be induced by DNA vaccine of pcDNA3.1(+)/Lpp20 in C57BL/6 mice, which might be helpful for further investigation concerning the immunoprotection of DNA vaccine.

Objective To explore the role of ADSCs and PRP in soft tissue defect repairing .Methods Harvest adipose tissue from inguinalis fat pad of SD rats ,isolation ,culture ,and identification the ADSCs through three differentiation method .Take 30 male SD rats about 6-7 weeks old randomly .Randomly selected 12 rats been take blood from heart .Preparation the PRP with modi-fied appel method .To count platelet of whole blood and PRP under microscope .Take the remaining rats .Divided the rat into 3 groups(n=6 in each group) randomly ,of which group A treatment with ADSCs and PRP ;Group B treatment with ADSCs ;Group C treatment whit PRP .Selected one side of skin defect wound for test randomly and the relative side skin defect is for control ,Handle all of the control wound to group D ;Observe the grow th of granulation tissue on the wound surface ;observe the inflammatory sur-rounding and the degree about epithelial .statistical analysis and record the wound size ,and calculation the shrinkage rate of wound in different periods .Record the time of completely healing time .Histologic observation of the wound healing tissue .Results Plate-let counting showed platelet of PRP is 5 .21 times than whole blood .The wound completely healed time :group A (18 .25 ± 1 .44 ) days ,group B(19 .13 ± 1 .28)days ,group C(19 .72 ± 0 .87)days ,group D(22 .31 ± 1 .65) days ,The time of each treatment group and control group was significantly obvious(P< 0 .05) .At 3 ,7 ,11 and 15 days after experimental treatment ,compared with the control group the experiment group of wound contraction rate was significantly obvious (P<0 .05) .Conclusion Application of AD-SCs with PRP can enhance the quality and shorten the wound healing time than used them alone .

Objective To study the effect of VacA on the secretion of THP-1 macrophages as an individual virulence determinant, and the effect of NF-kB on the secretion of THP-1 macrophages. Methods The recombinant plasmid pDsRed-Monomer-Cl/vacA was transfected into macrophages. The cytokine con-tent of TNF-α or IL-1β in the culture medium was tested quantitatively with ELISA kit, respectively. The content of NO or ROS in the culture medium was tested with Griess reagent or DCFH-DA fluorescent probe. The apoptosis rate of macrophages was tested by flow cytometry. The effect of PDTC, an inhibitor of NF-kB, on the secretion and apoptosis of macrophages transfected with the recombinant plasmids, was also studied. The activity of NF-kB was examined in THP-1 cells by electrophoretic mobility gel shift assay(EMSA). Re-suits At 6 h after transfection, the level of TNF-α and IL-1 β in macrophages transfected with the recombi-nant plasmids was significantly higher than that of the control group (P <0.05). At 6 h or 12 h after trans-fection, the level of NO and ROS in macrophages transfected with the recombinant plasmids was significantly higher than that of the control group (P <0.05). At 16 h after transfection, the apoptosis rate of macropha-ges transfected with the recombinant plasmids was significantly higher than that of the control group (P < 0.05). PDTC decreased the production of TNF-α, IL-1 β, NO, ROS and apoptosis rate induced by VacA. VacA was found to trigger NF-kB activation. Conclusion The over-expression of VacA fusion protein can up-regulate secretion and apoptosis of macrophages. Activation of NF-kB is probably involved in the produc-tion of TNF-α, IL-1β, NO, ROS and apoptosis induced by VacA.

Objective To investigate the immune response of mucosal immunization of new chitosan(CS) nanoparticles coating DNA vaccine. Methods The chitosan nanoparticles containing plasmid DNA encoding H. pylori lipoprotein Lpp20 gene were prepared using a complex coacervation method and then its speciality were analyzed. We then administered the naked plasmid DNA and chitosan-DNA nanoparticles to 6-week-old female BALB/c mice by intranasal or oral mucosal routes to observe the humoral and cellular immune responses. Results Naked plasmid pcDNA3.1 ( + )/Lpp20 and chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles both induced effective immune response in mice through mucosal vaccination. Specific IgG and sIgA antibodies of chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles groups were higher than that of naked pcDNA3.1 ( + )/Lpp20 group. The concentration of cytokines IFN-γ and IL-4 in cultural supernatant of T lymphocytes from chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles immunized mice increased greatly than that of control groups. After stimulated by corresponding antigen, the stimulation index of intranasal or oral delivery of chitosan-pcDNA3. 1 ( + )/Lpp20 nanoparticles group was significantly higher than that of pcDNA3.1( + )/Lpp20 group, CS group and PBS control group. Moreover, systemic and mucosal immune responses in mice induced by intranasal immunization were stronger than that of oral immunization. Conclusion Chitosan nanoparticles enhanced the immune response of pcDNA3.1 ( + )/Lpp20 DNA vaccine by intranasal or oral administration in BALB/c mice. Compared to oral administration, intranasal delivery of chitosan-pcDNA3.1 ( + )/Lpp20 DNA nanoparticles could induce stronger cellular and humoral immune responses in BALB/c mice.

ObjectiveTo screen a 12-mer phage display peptide library by the polyclonal antibody (pAb) against the recombinant adhesion protein of Mycoplasma genitalium (rMgPa) in order to obtain the antigenic mimic epitopes of MgPa.MethodsThe purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library.Seventy-four recombinant phage clones were randomly selected,and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX.The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA,competitive ELISA and Western blot analysis.Results After four rounds of biopanning,a significant enrichment of phages was achieved,the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences.Amongst 45 peptides,36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa.Immunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M.genitalium,P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I.Results of bioinformatics analysis by MIMOX demonstrated that S,A,F for cluster 1,A,K,I,T and L for cluster 2,K,S,L,R,D and I for cluster 3,may be the key consensus amino acid residues in the aligned mimotopes,respectively.ConclusionAntigenic mimics on MgPa were successfully identified and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent the immunodominant mimic epitopes of MgPa.And S,A,F K,I,T,L,R and D may be the key amino acid residues for the epitopes of MgPa.

Obiective To investigate the molecular characteristics of heteroresistant vancomycinintermediate Staphylococcus aureus(hVISA)in China and analyze the differences of the molecular characteristics between hVISA and VSSA(vancomycin-susceptible Staphylococcus aureus)isolates.Methods A total of 3 15 non-repetitive MBSA were collected from the national surveillance program in China in 2007.The isolates of hVISA were confirmed by modified population analysis profile-area under the curve(PAP-AUC).The genotypes of agr and SCCmec were determined by multiplex PCR,and spa typing was performed bv PCR and DNA sequencing.The pvl gene was detected bv PCR Results The prevalence of hVISA was 9.5%(30/315).Among 315 MRSA,SCCmec Ⅲ was the most popular type,which was found in 234 isolates(234/315,74.3%),followed by SCCmec Ⅱ,which was identified in 56 isolates (56/315,17.8%).The rate of SCCmec Ⅱ in hVISA(46.7%)was significantly hisher than in VSSA (14.7%,X~2=18.93,P＜0.001).The most prevalent agr type among 315 MRSA was agr 1 accounting for 73.6%(232/315).The agr 2 accounted for 18.7%(59/315),and agr 3 and agr 4 were very rare in clinical isolates.It was different in agr types between the two groups.The rate of agr 2 in hVISA(53.4%)was higher than in VSSA(15.1%).X~2 value was 26.08 and P value was less than 0.001 through X~2 test.There was a statistical significance in the result.There were 4 spa types in hVISA isolates,including t002 (13 isolates),t037(9 isolates),t030(6 isolates),and 1548(2 isolates).The pvl positive MRSA isolates were very low,accounting for 1.6%(5/315).Conclusions The prevalence of hVISA was relatively higher in China.Compared to VSSA,the majority(53.4%)of the hVISA strains were agr 2,which was obviously different from VSSA.hVISA isolates were more diverse by spa typing,

A molecularly imprinted two-dimensional photonic crystal hydrogel sensor was developed with erythromycin as imprinted molecule, polystyrene two-dimensional photonic crystal as templates, methanol as solvent, methacrylic acid as monomers and ethylene glycol dimethylacrylate as cross-linkers.The imprinted molecule was removed by methanol/acetic acid (9∶1, V/V).The results showed that the diameter of Debye ring increased 6 mm when the concentration of EM changed from 0 to 1×10-6 mol/L.Namely the lattice spacing decreased 30 nm.In addition, the diameter of Debye ring only increased 1.5 and 2.0 mm when the hydrogel immersed in 1×10-6 mol/L roxithromycin (RM) or erythromycin ethylsuccinate (EEs) solution.The result indicated that the sensor had high selectivity and could be used in determination of erythromycin with low cost and easy operation.