Objective:To evaluate dendritic cells induced immune response against hepatocellular carcinoma by pulsed CD34+ hematopoietic stem cells originated dendritic cells with p53 gene.Methods:CD34+ hematopoietic stem cells were harvested after mobilization by chemotherapy and G-CSF.CD34+ hematopoietic stem cell apheresis was induced to differentiate into dendritic cells by cytokine cocktail IL-4,GM-CSF and TNF-?.On day 7,dendritic cells were transfected with plasmid pEGFP-C3/p53 DNA.The CTL response triggered by p53 pulsed dendritic cells was assayed by MTT method.Results:Dendritic cells originated from CD34+ cell apheresis had typical dendritic stick and expressed high level CD1a,CD11c,CD80,CD86,and HLA-DR molecules.After being pulsed with p53 gene,dendritic cells expressed green fluorescence protein and immunofluorescence assay(Cy3 labeled anti-P53 antibody)showed that transfected dendritic cells emitted red fluorescence.Dendritic cells inducing CTL response against HMCC97 cells(P53 positive)and HepG2 cells(P53 negative)were assessed by MTT method.P53 pulsed dendritic cells could induce P53 specific immune response against HMCC97 cells and the cytotoxin rate was(49.3?4.6)% compared with pEGFP-C3 transfection group [(25.4?4.1)%] and control group[(24.8?3.8)%](P0.05).Conclusion:P53 gene transfecting hematopoietic stem cell apheresis originated dendritic cells could induce specific CTL response against P53-expressing hepatocellular carcinoma cells.P53 may be a potential candidate for dendritic cell based immunotherapy of cancer.

Objective To observe the effect of astragalus polysaccharides ( APS) on glucose homeostasis regulation and focus on glycogen synthase kinase 3 beta (GSK3 beta) activity and subcellular localization (nuclear translocation).Methods HepG2 human hepatoma cells were cultured in vitro and treated with high glucose of different concentrations (30, 40 mM) to induce hepatocyte endoplasmic reticulum stress model, then acquire optimum operating concentration.The HepG2 cells were treated with APS of different concentrations (50, 100, 200, 400 μg/mL) to select the most effective concentration.The HepG2 cells were divided into seven groups with different treatment: negative control group (C), positive control group (Tm), 30 mM high glucose-induced group (G30), 45 mM high glucose-induced group (G45), negative control+APS group (CA), positive control+APS group ( TA) and high glucose-induced+APS group ( GA).Effect of APS at different concentrations on proliferation activity of HepG2 cells were detected by MTT assay, transcription and shear levels of XBPlmRNA in HepG2 cells by quantitative real-time PCR, and phosphorylation levels of GSK3βin cytoplasm and nucleus by immunoblotting techniques.Results The optimum operating glucose concentration was 30 mM.The most effective APS concentration was 200μg/mL.The transcription and shear levels of XBPlmRNA in HepG2 cells of GA group were lower than those of G30 group ( P<0.05), respectively, but there were no significant differences between TA and Tm group.The phosphorylation levels of GSK3βin cytoplasm and nucleus of GA group were higher than those of G group(P<0.05), respectively, but there were no significant differences between TA and Tm group. Conclusion APS could improve hepatic steatosis, and its mechanism might be that APS inhibits the activity and nuclear localization of GSK3β, then alleviate endoplasmic reticulum stress.

Objective To clone a new human gene 5 trans-activated by pre-S1 protein of hepatitis B virus (HBV), PS1TP5, and explore its structure and function by bioinformatics analysis. Methods PS1TP5 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique by using HepG2 cDNA as template and inserted into pGEM-T vector by TA cloning. Recombinant eukaryotic expression vector pcDNA TM 3.1/myc-His A-PS1TP5 had been constructed by subcloning, followed by restriction enzyme digestion analysis and sequencing. Bioinformatic methods were used to analyze its possible physical and chemical characters, structure, and function. Results PS1TP5 was successfully amplified and cloned into pGEM-T and pcDNA TM 3.1/myc-His A vector by RT-PCR from HepG2 cDNA. The new gene had been confirmed by sequencing after PCR identification and restriction enzyme digestion and named as PS1TP5 because of its trans-active function. The sequence for the PS1TP5 gene had been deposited into GenBank, the accession number was AY427953. Bioinformatics analysis showed that its ORF was 438bp and translated a protein of 145 aa. Conclusion A new gene-PS1TP5 has been recognized, and its recombinant eukaryotic expression vector (pcDNA TM 3.1/myc-His A-PS1TP5) has been constructed. These results will certainly bring some new clues for the study of the biological function of new gene and pathogenesis of chronic hepatitis B.

Objective To study the exact function of HBeBP4A so as to investigate the gene expression of HBeBP4A in yeast cell.Methods Reverse transcription polymerase chain reaction(RT-PCR)was employed to amplify the gene of HBeBP4A from recombinant plasmids pcDNA 3.1/myc-HisA-HBeBP4A,and the gene was cloned into pGEM-T vector.The gene of HBeBP4A was cut from pGEM-T-HBeBP4A vector and cloned into yeast expressive plasmid pGBKT7,and pGBKT7-HBeBP4A was then transformed into yeast AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting hybridization.Results HBeBP4A gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The SDS-PAGE and Western blotting assay showed that the relative molecular weight of the expressed product was about 61.37kD,and HBeBP4A protein existed in yeast cells.Conclusion The findings suggested that HBeBP4A was successfully expressed into yeast system.

Mitral effective regurgitant orifice area was measured by using color Doppler flow convergenee method,and its value on quantilarian of mitral insufficiency Was evaluated.The results showed that the parameter was correlated well with regurgitant volume,regurgitant fraction,color Doppler jet beginning width and jet area(r=0.89、0.84、0.82 and 0.67 respectively,P<0.001～0,0001).The accurate rate in differentiating mild and moderate from severe mitral regurgitation was 95.7% with the effective regurgitant orifice area≥30mm2.The effective regurgitant orifie area is a good parameter in quantification of naitral insufficiency.

The value of flow convergence and jet parameters in quantification of mitral regurgitation was estimated.The resultS showed that there were excellent correlations between mitral regurgitant flow convergence volume and mitral regurgitant volume,regurgitant fraction(r=0.87～0.93,P<0.0001).The correlations between jet parameters and regurgtant volume,regurgitant fraction were different,jet beginning width:r=0.88 and 0.81(P<0.0001),jet area : r=0.72 and O.50 and 0.61(P<0.05)and the ratio of jet area to left atrial area:r=0.27 and 0.29(P>0.05).The accurate rates of regurgitant flow convergence volume and jet beginning width in differentiating the grades of mitral regurgitation were 91.3～95.7%and 82.6～95.7%respectively.Mitrai regurgitant flow convergence volume is an accurate and reliable parameter in the quantification of mitral regurgitation.Jet parameters are of relative value in semiquantitation of mitral regurgitation,especially the jet beginning width.

Flow convergence is a new method of color Doppler flow quantification.The effect of instrumental settings on flow convergence was investigated in an in-vitro model.The result showed that the flow convergence volume increased from 1.67±1.01 l/min to 2.20±1.35～2.30±1.43 l/min(P< 0.0001)when aliasing limit(same concept as Nyquist frequency)was changed from 23 cm/s to 12cm/s. There was no significant effect on flow convergence volume with the change of other instrumental settings,including Color gain,frame frequency,paket size,transmit power,transducer frequency,etc. Color Doppler flow convergence is influenced by aliasing limit.mainly because that the isovelocity surfaces in acceleration region developed by different aliasing limits are not always in the shape of hemisphere.The result suggested that besides the selection of aliasing limit fits to a hemispherical isovelocity surface,flow convergence is rarely affected by the other instrumental settings,and therefore is a relatively stable parameter of flow quantification.

Objective To estimate the anatomic variation of the right inferior phrenic artery(RIPA)with multi-detector tomography(MDCT)scans.Methods 45 patients with hepatocellular carcinoma(HCC)and 46 healthy subjects were examined by contrast-enhanced CT scan(CTA)at 16-section CT scanner.Then the images were reconstructed with MPR,VR and MIP.Results RIPA were detected by CTA in all cases(sensitivity was 100%).The origin of RIPAs directly from the aorta in 42%,celiac trunk in 37%,right renal artery in 15%,left gastric artery in 3% and proper hepatic artery in 2%.For the reconstructive images quality,MPR and MIP were better than VR,but in showing the origin of RIPAs,MPR and VR were better than MIP.In compared with normal group,the diameters of RIPAs in tumor group were larger.Conclusion MDCT estimates well for the anatomy of RIPAs,which is significant for planning and embolization of extrahepatic RIPA supply in HCC.

Blood Group Incompatibility ; complications ; Erythroblastosis, Fetal ; etiology ; Humans ; Infant, Newborn ; MNSs Blood-Group System ; immunology ; Male

OBJECTIVE To investigate the prevalence of strains producing extended-spectrum ?-lactamases (ESBLs) and AmpC ?-lactamases in Pseudomonas aeruginosa,and to supply the laboratory evidence for antibiotic rational application in clinic. METHODS Totally 105 clinical isolates of P. aeruginosa were identified with VITEK-32. Drug sensitivities were determined by Kirby-Bauer methods. ESBLs were detected by double-disc synergy test,and cefoxitin three dimensional test was applied to filter AmpC positive strains. RESULTS Among 105 strains of P. aeruginosa,28 strains (26.7%) were AmpC enzymes positive,20 strains (19.0%) were ESBLs positive,and 5 strains(4.8%)were AmpC+ESBLs positive. The detective rate of producing AmpC ?-lactamases strains was higher than that of producing ESBLs strains. There was significant difference between them. CONCLUSIONS ESBLs and AmpC ?-lactamases are two main enzyme types conferring resistance to ?-lactam antibiotics in clinical isolates of P. aeruginosa. Imipenem can be the first choice for treating infections caused by P. aeruginosa producing ESBLs or AmpC ?-lactamases.