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.

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

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 construct a luciferase gene expression vector containing full-length 3' untranslated region(3'UTR)of mouse vascular endothelial growth factor-C(VEGF-C)gene,and to observe the effects of VEGF-C 3'UTR on luciferase gene expression by a double-fluorescence report system.Methods Polymerase chain reaction(PCR)was used to amplify VEGF-C 3'UTR and a 312bp VEGF-C coding region(CR)fragment from full-length VEGF-C cDNA in mouse Lewis lung cancer cells.The luciferase expression vectors containing VEGF-C 3'UTR or VEGF-C CR were constructed by subcloning the PCR products to luciferase reporter vector pGL3-Promoter using gene engineering technology,and then they were transfected to mouse Lewis lung carcinoma cells by LipofectamineTM 2000.The activities and mRNA expression of luciferase were detected by Dual-Luciferase Reporter System and quantitative RT-PCR,respectively.Results Mouse VEGF-C 3'UTR(429bp)and VEGF-C CR(312bp)were successfully amplified by PCR.The VEGF-C 3'UTR and VEGF-C CR fragments were successfully inserted into pTA2 vector,and then subcloned to pGL3-Promoter vector at Xba Ⅰ site by using restriction endonucleases analysis.The DNA sequences and insertion orientation of PCR products were all correct by sequencing analysis.The resulted luciferase expression plasmids were named pGL3-VEGF-C 3'UTR and pGL3-VEGF-C CR,respectively.Dual-Luciferase Reporter System detection and quantitative RT-PCR showed that in Lewis lung carcinoma cells,the activities of luciferase and expression of luciferase mRNA in the pGL3-Promoter group were higher than that in the pGL3-VEGF-C 3'UTR group,and there was no significant difference between pGL3-VEGF-C CR group and pGL3-Promoter group.Conclusion VEGF-C 3'UTR can inhibit luciferase gene expression.

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.

OBJECTIVE To study resistance of Enterococcus in clinical isolates to high-level gentamicin(HLG) and investigate drug resistance of these Enterococcus.METHODS Totally 140 strains of Enterococcus were detected resistance to antibiotics by Kirby-Bauer test and agar-screening test.Data were analyzed using WHONET5.3 software.RESULTS Same result was obtained by two kinds of detecting methods,there were 73 strains of high level gentamicin resistance(HLGR,52.1%) and 67 strains of non-HLGR(47.9%),the resistance rate of HLGR was higher than that of non-HLGR,the isolation rate of vancomycin resistanct Enterococcus was 3.6%.CONCLUSIONS The most common enterococci causing hospital infection are E.faecalis and E.faecium.The drug resistance rate of E.faecium is higher than that of E.faecalis.It is imperative to select the proper method to detect HLGR of Enterococcus,and select proper antibiotic in terms of antibiotic susceptibility test during clinical therapy.

Objective The aim of this study was to assess the clinical value of pro-gastrin releasing peptide (ProGRP) , squamous cell carcinoma antigen (SCC-Ag), cytokeratin 19 fragment antigen 21-1 (Cyfra21-1) and carcino-embryonic antigen (CEA) in the diagnosis and clinical stage of lung cancer in Chinese patients.Methods Patients with lung cancer and benign lesions confirmed by pathology were enrolled in Peking Union Medical College Hospital from January 2013 to October 2014.The serum levels of four tumor markers (ProGRP, SCC-Ag, Cyfra21-1 and CEA) were measured using immunoassays before treatment.The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the areas under the receiver operating characteristic curve (AUCROC) of these four tumor biomarkers were analyzed for the diagnosis of lung cancer.Results A total of 134 patients were finally analyzed, including 73 patients with lung cancer and the other 61 patients with benign lung disease.The diagnostic sensitivity of serum Cyfra21-1 to lung cancer was 67.1％, the specificity 45.1％, the AUCROC 0.658.The diagnostic sensitivity of the panel including ProGPR, Cyfra21-1 and CEA to lung cancer was 75.3％ , the specificity 57.4％ , the AUCROC 0.702.In the lung cancer group, the AUCROC of ProGRP over 65 ng/L to diagnose small cell lung cancer was 0.954;the AUCROC of SCC-Ag over 1.5 μg/L to diagnose squamous cell lung cancer was 0.788;the AUCROC of Cyfra21-1 to diagnose non-squamous-non-small-cell lung cancer was 0.716.In small cell lung cancer patients, the level of ProGRP in limited-disease small cell lung cancer (LD-SCLC) were significantly higher than that in extensive-disease small cell lung cancer (ED-SCLC) (P =0.005).Conclusion This panel of serum tumor markers including ProGRP, Cyfra21-1 and CEA improves the diagnostic specificity and sensitivity in patients with high-risk lung cancer.The serum CEA level of advanced lung cancer patients is significantly increased.The high level of serum ProGRP predicts the ED-SCLC.

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.

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.