ObjectiveTo generate a prokaryotic expression system of series predominant epitopes (UreB322 and UreB527) of Helicobacter pylori UreB protein,and to synthesize a multiple antigenic peptide (MAP) vaccine by linking both the two epitopes with a peptide carrier (Poly-Asp-Lys),and to determine the immunogenicity and immunoprotection of the MAP vaccine.MethodsLinking primer PCR was performed to generate an enterokinase(EK) site-containing series UreB322 and UreB527 epitope encoding gene for construction of its prokaryotic expression system.The expressed target recombinant fusion protein 8 ×[rEK-UreB322-EK-UreB527-EK] was hydrolyzed with EK and then rUreB322-EK and rUreB527-EK epitope peptides were extracted using a Sephadex G-25 column.rUreB322-EK,rUreB527-EK and Poly-Asp-Lys were linked using carbodiimide method to produce a MAP vaccine (MAP-rUreB322/B527).The antigenicity and immunoreactivity of each of the two epitope peptides and MAP-rUreB322/527 were determined by ELISA and Western blot assay.An animal H.pylori strain SS1-infected model in BALB/c mice was used to detect the immunoprotection of MAP-rUreB322/527.ResultsAn octuple-repeated series UreB322-UreB527 encoding gene and its prokaryotic expression system were obtained.The yield of target fusion protein 8×[rEKUreB322-EK-rUreB527-EK] was as high as 48％ of the total bacterial proteins.EK hydrolyzed the target fusion protein completely into rUreB322-EK and rUreB527-EK peptides.The linking ratio of rUreB322-EK,rUreB527-EK and Poly-Asp-Lys was as high as 92.5％.The antibody against whole cell of H.pylori and rUreB-IgG could recognize and combine with the rUreB322-EK,rUreB527-EK or MAP-rUreB322/527.The specific serum antibody level in MAP-rUreB322/527-immunized mice was significantly higher than that in rUreB-immunized mice (P＜0.05).The immunoprotective rates(83.3％ and 91.7％ ) by immunization with50 or 100 μg MAP-rUreB322/527 in the H.pyloristrain SSI-infected mice were significantly higher that those(d1.7％ and 50.0％ ) by immunization with equal rUreB(P＜0.05).ConclusionAn gene composed for encoding a repeated series predominant epitopes of H.pylori UreB protein and its prokaryotic expression system are successfully generated in this study.MAP-rUreB322/527,the multiple antigenic peptide vaccine based on the two predominant epitopes of UreB,can noticeably increase the immunoprotection in H.py/or/infected mice.

Objective To systematically compare propofol and isoflurane for myocardial protection in patients undergoing coronary artery bypass grafting (CABG).Methods Electronic databases were searched for randomized controlled clinical trials comparing propofol and isoflurane for myocardial protection in patients undergoing CABG.Data which were extracted independently by two reviewers included the general data of patients,premedication,induction of anesthesia and anesthetics applied during maintenance of anesthesia,level of cardiac troponin I (cTnI) before operation and at 6,12,24 and 48 h after operation,requirement for positive inotropic agents during operation,and development of myocardial infarction within 24 h after operation.Meta-analysis was conducted using Review Manager 5.0.2.Results Sixteen randomized controlled clinical trials involving 794 patients were included in this meta-analysis.The patients were divided into 2 groups:propofol group (n =405) and isoflurane group (n =389).There were no significant differences between the two groups in the plasma concentration of cTnI after operation,incidence of myocardial infarction within 24 h after operation,and requirement for positive inotropic agents during operation (P ＞ 0.05).Conclusion There is no significant difference between propofol and isoflurane for myocardial protection in the patients undergoing CABG.

Objective To generate a prokaryotic expression system of Salmonella paratyphi A nmpC gene that encoding an outer membrane protein(OMP),and to determine immunogenicity and immonuprotection of the recombinant expressed product rNmpC and carrying and expression frequencies of the nmpC genes in isolates of S.paratyphi A.Methods A nmpC gene clone was obtained from a clinical S.paratyphi A strain JH01 by PCR and T-A cloning method,and then a prokaryotic expression system of the gene clone was generated.SDS-PAGE and Bio-Rad Agarose Image Pattern Analysis System were applied to examine the expression and yield of rNmpC.Antigenicity and immunoreactivity of rNmpC were determined by immunodiffusion test,Western blot assay and micro-Widal's test.The carrying and expression rates of nmpC genes in 98 S.paratyphi A isolates were detected by PCR and ELISA.By a mouse infection model,the immunoprotective effect of rNmpC against the lethal challenge of S.paratyphi A was determined.Results All the cloned nmpC genes had 100% nucleotide and putative amino acid sequence identities compared to the reported sequencing data.The expression yield of rNmpC was approximately 30% of the total bacterial proteins.rNmpC could efficiently induce rabbits to produce specific antibody and present positive Western hybridization signals with S.paratyphi A antiserum.All the tested S.paratyphi A strains have nmpC gene as well as express NmpC protein,but no nmpC gene could be detectable in S.typhi,S paratyphi B and S paratyphi C.Immunization with 100 μg and 200 μg rNmpC contributed 41.7%(5/12) and 66.7%(8/12) immunoprotective rates in mice,respectively.The sera from rNmpC immunized mice and survival mice in the NmpC is an unique OMP antigen of S.paratyphi A with conserved sequence,extensive distribution and natural expression.This OMP can be used as one the candidate antigens for developing multiple-valence genetic engineering vaccine of S.paratyphi A based on its fine immunogenicity and certain immunoprotection.

Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.

In total, 106 patients with maintenance hemodialysis (MHD) were divided into two groups based on their valsartan administration, and 53 healthy controls were recruited in the study. Plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), resistin and high sensitivity C-reactive protein (hs-CRP) were measured by enzyme-linked immunosorbent assay (ELISA) before and after valsartan treatment. Plasma levels of resistin, IL-6, TNF-α and hs-CRP were all significantly increased in patients with MHD as compared to those in healthy controls (P＜0. 05). Among patients with MHD the plasma levels of resistin, IL-6 and hs-CRP in valsartan group were lower than those in non-valsartan group (P＜0. 05).

BACKGROUND:Astragaloside Ⅳ is a major component of Huangqi,promoting proliferation and differentiation of bone marrow mesenchymal stem cells;however,the mechanism has been less reported yet.OBJECTIVE:To explore the effect of Astragaloside Ⅳ on expression of multiple hematopoietic growth factors in bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells were isolated from adult Wistar rats by using the method of adhesive culture and clone,and they were then plated on 96-well plate and separately incubated with 100 uL Astragaloside Ⅳ(25,50,100,200 g/L)for 72 hours.The cells in the control group were cultured with an equal volume of DMEM-LG culture liuquid.Indirect immunofluorescence was used to detect the biological activity,MTT method was used to evaluate the effect of Astragaloside Ⅳ on proliferation and differentiation of bone marrow mesenchymal stem cells,and RT-PCR method was used to measure the expression of hematopoietic growth factors in bone marrow mesenchymal stem cells.RESULTS AND CONCLUSION:The 3~(rd)-passage bone marrow mesenchymal stem cells highly expressed CD44 but lowly expressed CD45.As compared with control group,Astragaloside Ⅳ promoted proliferation of bone marrow mesenchymal stem cells in a time/dosage-dependent manner,in particular,the 200 g/L Astragaloside IV and 72-hour intervention(P＜ 0.05).SCF expression was significantly increased in the drug group compared with control group(P ＜ 0.01);however,TPO,GM-CSF,and TGF-β1 expressions were not changed significantly(P ＞ 0.05).Moreover,interleukin-3 expression was not found in the bone marrow mesenchymal stem cells.Astragaloside Ⅳ promoted in vitro proliferation of bone marrow mesenchymal stem cells,possibly involving in SCF secretion.

Objective To investigate the changes of zinc in maternal plasma and MMP-9,collagen Ⅳ levels in serum and matrix metalloproteinases-9 (MMP-9) level in amniotic fluid in patients with premature rupture of membranes at term(TPROM).Methods Thirty cases who were diagnosed as PROM at term during Nov.2012 to Mar.2013 were enrolled as case group,and 30 cases delivered during the same time without PROM were enrolled as control group.Maternal blood and ammiotic fluid were collected from all the cases.The level of zinc in maternal plasma was measured by atomic absorption method and the levels of MMP-9 in serum and amniotic fluid were detected by enzyme linked immunosorbent assay(ELISA),while the level of collagen Ⅳ in serum was measured by up-conversion luminescence method.The relationship among them was analyzed.Results Compared to control group,there were statistically significant difference between TPROM and control groups in terms of the level of zinc,collagen Ⅳ,MMP-9 in serum and MMP-9 in amniotic fluid (zinc:(109.10 ± 16.07) μmol/L vs.(90.54 ± 10.99) μmol/L; t =-5.22,P ＜ 0.001 ; collagen Ⅳ:(56.86 ±41.26) μg/L vs.(88.61 ±44.87) μg/L;t =2.852,P =0.006;MMP-9 in serum:(1 463.25 ±483.6) μg/L vs.(1 196.9 ± 357.43) μg/L,t =-2.426,P =0.018 ; MMP-9 in amniotic fluid:(125.48 ± 67.18) μg/L vs.(72.64 ± 60.74) μg/L,t =-2.873,P =0.006).Zinc level in maternal plasma and collagen Ⅳ in serum had a negative relationship in TPROM (r =-0.261,P =0.044).Zinc level in maternal plasma and MMP-9 level in serum had a positive relationship in TPROM (r =0.274,P =0.034).MMP-9 levels in serum and amniotic fluid had a positive relationship in TPROM (r =0.264,P =0.047).There were no significant relationship between zinc level in maternal plasma,MMP-9 level in amniotic fluid,collagen Ⅳ and MMP-9 levels in serum,collagen Ⅳ in serum and MMP-9 in amniotic fluid (r =0.215,-0.172,-0.172 ; P ＞ 0.05).Conclusion The level of zinc in maternal plasma and increase of MMP-9 in serum and amniotic fluid of women and decrease of the level of collagen Ⅳ in serum are related to the occurrence of TPROM.

Objective To study the inhibitory effects of dehydroepiandrosterone (DHEA) and its metabolites-dehydroepiandrosterone sulfate (DHEAs) on the proliferation of HepG2 and HT-29 and their mechanism.Methods HepG2 and HT-29 were incubated by DHEA or DHEAs with different concentrations (1,10,50,100 and 200 ?mol?L-1) for 8,24,48,72 h and routine culture was used as control.The inhibitory rate was detected by using MTT chromometry and BrdU assay respectively.The activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR),glucose -6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) were examined simultaneously.Results ①MTT chromometry:DHEA with different concentrations obviously inhibited the growth of HepG2 and HT-29 cells compared with control group(P0.05).②BrdU assay:the growth of cells were significantly inhibited by DHEA with concentrations of 50,100 and 200 ?mol?L-1,especially to HepG2 cells(P0.05).Conclusion DHEA has strong anti-proliferative effects on both HepG2 and HT-29 cell lines and inhibitory effects on the activities of G6PD or HMGR,however,DHEAs has no obvious effect.

Objective: To determine plasma levels of soluble tumor necrosis factor-related apoptosis inducing ligand (sTRAIL) and its soluble death receptor (sDR4, sDR5) in essential hypertension (EH) patients with left ventricular hypertrophy (LVH). Methods: Enzyme linked immunosorbent assay (ELISA) was used to measure plasma levels of sTRAIL, sDR4 and sDR5 in 50 EH + LVH patients (EH + LVH group), 50 EH patients without LVH (EH group) and 50 healthy subjects (healthy control group), and the results were compared and analyzed among three groups. Results: ① Compared with healthy control group and EH group, there were significant increase in plasma levels of sTRAIL [(0.95±0.11) ng/ml vs. (1.12±0.86) ng/ml vs. (1.74±1.19) ng/ml], sDR4[(2.38±0.32) pg/ml vs. (5.63±1.05) pg/ml vs. (8.72±1.14) pg/ml] and sDR5[(< 6 pg/ml) vs. (39.19±8.23) pg/ml vs. (78.21±11.2) pg/ml] in EH + LVH group, P<0.01 all; and levels of sDR4 and sDR5 in EH group were significantly higher than those of healthy control group (P<0.01 both), but there was no significant difference in sTRAIL level between the two groups (P>0.05); ② Pearson correlation analysis indicated that there were significant positive correlation among levels of sTRAIL, sDR4 and sDR5 in EH + LVH patients (r=0.325~0.410, P<0.05 or <0.01). Conclusion: Plasma levels of sTRAIL, sDR4 and sDR5 may be valuable indexes for prediction of left ventricular hypertrophy in patients with hypertension.

Aim To obtain the HCC cell lines which could coexpressed the B7.1 and SEA. Methods The positive clones expressing the B7.1 and SEA were screened by immunohistochemical staining. The amount of SEA in culture supernatant was detected by ELISA. Results HCC cell clones coexpressing B7.1 and SEA were obtained, and expression amount of SEA in culture supernatant reached 10～ 14× 10-8g/L. Conclusion The co-rec-ogenition immune effective system of SEA and B7.1 on HCC cells is established.