Objective:To construct secreted chimeric vector of AFP-HSP70 and investigate transient expression of vectors in eukaryotic cells.Methods:Vectors were constructed by routine molecular technique.Fusion genes were linked with G-S-G-G-S linker.Vectors were transfected into COS-7 cells by Lipofectamine 2000. Proteins were assayed by immunocytochemistry and RT-PCR for its RNA.Results:Proteins expressed in COS-7 were comfirmed by immunocytochemistry 48 hours later and RNA was detected by RT-PCR.Conclusion:AFP-HSP70 chimeric vector is constructed successfully and could be expressed in eukaryotic cells.

Objective To study the HBsAg specific CTL activities activated by dendritic cells derived from human monocytes on the HepG2/S target cells, and further to probe into the anti HBV effect of HBsAg DC (dendritic cell) vaccine. Methods DCs are proliferated from human peripheral blood monocytes by adding GM CSF and IL 4 and then HBsAg specific CTL are activated by DCs pulsed by HBsAg; The target cell line (HepG2/S) expressing HBsAg was set up by transfecting recombinated plasmid with HBV/S gene (PLXSN/S) into HepG2/S cell line; HBsAg specific CTL and HepG2/S target cells were cocultured in 96 well flat bottomed microtiter plates for 48 hours at 37 ?C in 5% CO 2, and then HBsAg specific CTL activities activated by DCs pulsed by HBsAg were detected by counting the number of killed target cells. Results HBsAg specific CTL activated by dendritic cells derived from human monocytes could produce strong killing effect on the target cells HepG2/S cells. It's specific CTL activities were 3.8%, 69.5% and 85.1% in different concentration (0 ?g/L,50 ?g/L and 100 ?g/L) respectively. While, it had no killing effect on the HepG2 cells, so the HBsAg specific killing effect was specific. Conclusions The result shows that HBsAg specific CTL activated by dendritic cells derived from human monocytes has strong killing effect on the HBV.

Objective To analyze and monitor the distribution of EBSLs-producing E.coli in our hospital and its resistance to commonly used antibacterial drugs.Methods The drug sensitivity test results of E.coli cultured in our hospital from 2008 to 2011 were continuously observed and performed the summary and the descriptive analysis.Results The detection rate of EBSLs-produ-cing E.coli during these 4 years was more than 50%.The generation rate of ESBLs-producing E.coli from the pharyngeal swab samples was the highest.The drug resistance of EBSLs-producing E.coli was mostly higher than that of non-EBSLs-producing E. coli,the difference was statistically significant(P <0.05).EBSLs-producing E.Coli showed the multi-drug resistant phenomenon. But the resistance rate of EBSLs-producing E.Coli to some antimicrobial drugs had the decreasing tendency year by year.Conclusion The drug-resistance situation of ESBLs-producing E.Coli is serious.The diceovered carbapenems-resistance ESBLs-producing E. Coli should cause the concern.The antibacterial drugs with increased drug-resistance rate should be replaced by the antibacterial drugs with the gradually decreased drug resistance rate.Strengthening the bacterial drug resistance minitoring can timely discover the change trend of clinically isolated bacteria and has the improtant significance to provide reference for clinically empirical medica-tion.

Antiviral Agents ; therapeutic use ; Carcinoma, Hepatocellular ; drug therapy ; virology ; Hepatitis B virus ; Humans ; Liver Neoplasms ; drug therapy ; virology ; Nucleotides ; therapeutic use

Objective To explore the effect of nucleos(t)ide analog (NA)antiviral treatment on the pathological differentiation of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC)and the prognostic factors of HCC.Methods Totally 127 patients with HBV-related HCC who were hospitalized and received partial hepatectomy in First Affiliated Hospital of Harbin Medical University from March 2007 to November 2013 were included in this study.Sixteen cases received antiviral treatment before operation and the remaining 111 cases had no history of NA treatment.The differences of histopathological grading were compared between the two groups.Twenty-nine patients received antiviral treatment for the first time after surgery,and the rest 82 patients did not.All these patients were followed up for survival and recurrence.Multivariate analysis was used to explore the prognostic factors for HCC.The categorical variables were analyzed byχ2 test or Fisher exact test.Survival rate was compared with Log-rank test. Univariate or multivariate Cox regression analysis was used to explore the related factors of survival. Results The proportions of well-,moderately- or poorly-differentiated HCC in patients with antiviral treatment before surgery were 18.75 %,68.75 % and 12.5 %,respectively.Whereas the proportions in those without treatment were 16.22%,66.67% and 17.11 %,respectively.There was no significant difference in histopathological grading of HCC between the two groups (χ2=0.224,P =0.885 ).The overall median survival time was 39 months.The 6-month,1-and 2-year survival rates were 91 .7%, 77.5 % and 59.3%,respectively.The 6-month,1- and 2-year survival rate of postoperative antiviral treatment were 96.3%,92.4% and 78.5 %,respectively,which were significantly higher than those of no antiviral treatment group (85 .9%,70.0% and 48.5 %,respectively;χ2= 6.967,P = 0.008 ). Univariate analysis showed that tumor number,size,portal vein transfer,AFP level,postoperative antiviral treatment,histopathological grading,TNM staging,BCLC staging,γ-GT and PTA were prognostic factors for postoperative HCC survival.Multivariate analysis showed that AFP level (HR=1 , 95 %CI :1 .0004—1 .002,P =0.004),postoperative antiviral treatment (HR =0.38,95 %CI :0.38—0.15 ,P =0.04)and BCLC stage (B vs A:HR=1 .55 ,95 %CI :0.76—3.18;C vs A:HR=3.63,95 %CI :1 .31 —10.09,P =0.04)were independent prognostic factors.Conclusions Preoperative antiviral treatment has no impact on the histopathological grading of HCC. BCLC stage, AFP level and postoperative antiviral treatment are independent prognostic factors for HBV-related HCC.

Objective To investigate the effects of human S100A6 on ?-catenin in human osteosarcoma cell lines MG63 and U2OS.Methods Cell lines MG63 and U2OS were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene,AdS100A6 and AdSiS100A6 respectively,to up-regulate and down-regulate the expression of S100A6. Then RT-PCR,Western blot and immunocytochemistry were used to detect mRNA and protein (level and/or distribution) of ?-catenin.Results In both cell lines,with up-regulated S100A6,expression of ?-catenin mRNA and protein increased(P

Objective To investigate the effects of human S100A6 on β-catenin in human osteosarcoma cell lines MG63 and U2OS. Methods Cell lines MG63 and U2OS were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene, AdS100A6 and AdSiS100A6 respectively, to up-regulate and down-regulate the ex-pression of S100A6. Then RT-PCR, Western blot and immunocytochemistry were used to detect mRNA and protein (level and/or distribution) of β-catenin. Results In both cell lines, with up-regulated S100A6, expression of β-catenin mRNA and protein increased(P <0. 05) and β-catenin protein increase was more obvious in nuclear than in cytoplasma; while down-regulating S100A6, both the mRNA and protein level of β-catenin decreased (P<0. 05) ; β-catenin protein decrease was more obvious in nuclear than in cytoplasma, too. Conclusion In-creasing Wnt/β-catenin signaling activity may be a mechanism that S100A6 involves in tumor development.

Objective The biological effects of BMP3 on osteosarcoma were investigated by treating human osteosarcoma cell lines MG63,and U2OS with human BMP3(hBMP3).Methods Osteosarcoma cells in experimental groups were respectively treated with AdBMP-3 and rhBMP3-CM,control groups with AdGFP and rGFP-CM,the blank group with neither.Their ability of proliferation,apoptosis,transmigration and differentiation were respectively detected by trypan blue exclusion test,terminal deoynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) and acridine orange-ethidium bromide fluorescent stain(AO/EB),transwell-room and alkaline phosphatase(ALP) activity reagent kit.Results(1) All the indexes detected were not significantly different between two control groups.(2) Compared with control groups,the cell survival rate showed a significant decrease in experimental groups.(3) The apoptosis indexes increased.(4)The number of trans-membrane cell decreased.(5)The activity of alkaline phosphatase increased after treatment with AdBMP3 and rhBMP-3 for 3 days in MG63,5 days in U2OS.Conclusion hBMP3 inhibit osteosarcoma cells growth and promote bone formation.

OBJECTIVETo study the HBsAg transient expression in HepG2 or COS-7 cells with eukaryotic expression plasmids inserting HBsAg gene (pCI-S and pcDNA3.1-S) and the efficacy of naked DNA immunization in mice.

METHODSFirstly, the recombinant plasmids of pCI-S and pcDNA3.1-S were constructed by the cloning technique and the accuracy of these constructs was confirmed by restriction enzyme digestion and DNA sequencing. Secondly, plasmids of pCI-S and pcDNA3.1-S were transferred into HepG2 and COS-7 cells, respectively by means of cationic liposome. HBsAg transient expression was assayed by ELISA in cell culture supernatants and cell lysates. Thirdly, plasmids were injected into quadriceps muscles of BALB/C mice and serum samples were obtained from individual immunized or control mice 4 weeks after injection and boost injection, respectively. Anti-HBs were assayed in mice sera by ELISA. HBsAg-specific CTL responses of spleen cells from immunized mice were tested by the LDH method.

RESULTSPlasmids of pCI-S and pcDNA3.1-S allowed HBsAg transient expression in cell culture supernatants and cell lysates of HepG2 or COS-7 cells. Intramuscular immunization of BALB/C mice with plasmids of pCI-S or pcDNA3.1-S elicited the antibody and cytotoxic T lymphocyte responses to HBsAg.

CONCLUSIONSThe vectors used in this study are effective to induce prime antibody and HBsAg-specific-cytotoxic T lymphocyte responses to HBsAg in mice after intramuscular immunization.

Animals ; COS Cells ; Cloning, Molecular ; DNA, Viral ; genetics ; Eukaryotic Cells ; metabolism ; Female ; Gene Expression ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis, Viral, Animal ; immunology ; prevention & control ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Cells, Cultured ; Vaccines, DNA ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

The construction of featured specialties is the current development strategy of community health service institutions to improve the service scope and to meet the health needs of residents. The rehabilitation medicine has undergone 12 years of development and become a relatively mature featured specialty in Fenglin Community Health Service Center. Based on the Fenglin′s experience, this article discusses the development status and restriction bottlenecks of general practice, and the development status and trend of rehabilitation medicine in the community; and also explores the integrated development model of community-featured specialty with general practice.