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 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.

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

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.

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 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 evaluate the morphological and signal intensity changes after anterior cruciate ligament reconstruction , and to analyze the correlations between MRI evaluation and clinical examination .Methods Totally 34 patients who underwent anterior cruciate liga-ment reconstruction in our hospital from July 2013 to June 2015 were given MRI examination .The morphology ,orientation and signal intensity of the anterior cruciate ligament grafts were evaluated on axial ,sagittal and coronal images .The grafts were assessed with Rak method ,and the clinical assessment was adopted with physical assessment method ,including drawer test ,axial shift test and Lachman test .The correlations be-tween MRI evaluation and clinical examination results were further analyzed by SPSS 16.0.Results After anterior cruciate ligament recon-struction,79%of the graft were visualized as a smoothly continuous low signal (well-defined type),15%of the graft showed increased signal intensity with only a small part of low signal bands ( intermediate type ) ,and 6% of the grafts showed remarkably increased signal intensity (indiscernible type).A statistically significant correlation was identified in MRI evaluation and clinical examination results (P<0.05). Spearman’ s correlation coefficient was 0.747 2 which indicating significantly positive correlation .Conclusion Magnetic resonance imaging is a noninvasive high-resolution imaging technology and it is an effective tool for evaluating anterior cruciate ligament reconstruction .

Objective To study the clinical efficacy and safety of Silybin-nanosuspension in the treatment of liver cancer.Methods 56 patients with liver tumor were collected from February 2014 to May 2015 in our hospital,and all patients were randomly divided into treatment group(n=24)and control group(n=32).The treatment group was treated with oral Silybin-nanosuspension 360mg(Silybin)and control group was treated with Silybin capsule 360mg(Silybin),once a day.The treatment was over once the following conditions appear,the disease progression,or intolerable toxicity,or the lesion site can be treat with surgery,or patient death.Evaluating the efficacy through comparing the data of objective response rate,disease control rate, progression-free survival and overall survival,and record the adverse reactions through measuring the values of indicators of blood toxicity ( leukocytes, neutrophils,platelets and hemoglobin ) and the liver function parameters ( Valley alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) .Results The objective response rate, disease control rate, progression-free survival and overall survival of treatment group were significantly better than control group(P<0.05),and the parameters of blood toxicity and liver function were no significant differences.Conclusion The Silybin-nanosuspension is safe and effective for the treatment of liver malignancies.

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.