Objective To explore origin recognition complex 1(ORC1) expression in the rat vascular muscle cells at different phases of proliferation.Methods Vascular smooth muscle cells(VSMCs) of thoracic aorta of rats in primary culture were obtained by the adherence method of tissue culture.Total RNA of VSMCs was extracted.The expression of ORC1 mRNA of VSMCs at different phases of proliferation was determined by reverse transcription polymerase chain reaction(RT-PCR) and the expression of ORC1 protein by immunocytochemistry and laser confocal microscopy.Results Cultured VSMCs were confirmed by light microscope and immunocytochemistry.The expression of ORC1 mRNA in the quiescence stage of VSMCs was not found.After VSMCs were stimulated with serum,the level of ORC1mRNA had an obvious increase at 6 h,peaked at 12 to 24 h and decreased in the following 24 h.The expression of ORC1 protein was also not found in the quiescence stage of VSMCs,but the level of ORC1 protein during proliferation of VSMCs was significantly increased.Conclusion ORC1 may have an important role during the process of VSMCs proliferation in rats.

Objective To explore the expression of origin recognition complex1(ORC1) during DNA replication progress of rat vascular muscle cells(VSMCs).Methods VSMCs of rat thoracic aorta were obtained by the adherence method of tissue culture.The growth curve was drawn by MTT.The association between DNA replication and the expression of ORC1 mRNA and protein in different growth phases of VSMCs was analyzed.Results The expression of ORC1 mRNA and protein in quiescence stage of VSMCs was not found.After stimulated with serum,the expression of ORC1 mRNA in rat VSMCs increased significantly,peaked at 12-24 h.The expression of ORC1 protein was similar to ORC1 mRNA in VSMCs.Meanwhile,the higher DNA replication of stimulated VSMCs was observed,peaked at 12-24 h after serum addition. Conclusion ORC1 may be involved in the DNA replication of rat VSMCs during the progress of proliferation.

Objective To investigate the influence of RNA interference targeting ORC1 gene on the proliferation of rat vascular smooth muscle cells(VSMCss).Methods VSMCss were transfected with siRNA targeting ORC1 gene by liposome.The expression of ORC1 protein was detected by Western blotting.MTT test and ~(3)H thymidine~()(~(3)H-TdR) incorporation were used to detect VSMCss proliferation.The expression of proliferating cell nuclear antigen(PCNA) in VSMCss was detected by immunocytochemistry.Results After transfected of the three pairs of siRNA targeting ORC1 genes respectively,the expression of ORC1 was all lower than that in the control group(non-transfection and negative siRNA).Especially,that in the group transfected of the second pair positive siRNA decreased most significantly.The optical density of MTT,~(3)H thymidine incorporation and the expression of PCNA decreased significantly in VSMCss transfected with siRNA targeting ORC1 gene as compared with that in the control group.Conclusion ORC1 gene silenced by RNA interference can inhibit VSMCss proliferation.

Interferon-induced Transmembrane Proteins (IFITMs) were identified through small interference RNA (siRNA) screening method in 1980s. The antiviral properties of the IFITMs were firstly discovered in 1996. Recently, its antiviral effect and mechanism have become a research hotspot. Many studies have shown that IFITM can inhibit the replication of multiple pathogenic viruses, including influenza A virus (IAV), Human Immunodeficiency Virus (HIV-1), hepatitis C virus (HCV), Ebola virus (EBOV), West Nile virus and so on. IFITMs inhibit the replication of virus in the early stage of the viral life cycle, which occurred before the release of viral genomes into the cytosol. Recent studies indicate that IFITM proteins could block viral replication by mediate viral membrane fusion. However, the mechanism is still under investigation. Here we review the discovery and characterization of the IFITM proteins, elucidate their antiviral activities and the potential mechanisms.
Animals ; Humans ; Interferons ; genetics ; immunology ; Membrane Proteins ; genetics ; immunology ; Virus Diseases ; genetics ; immunology ; virology ; Viruses ; genetics ; immunology

The microencapsulated genetic cells may be a new platform instead of genetic engineering drugs, as they can overcome the genetic engineering drugs' shortages such as short half-life in vivo, low activity, and incomplete elimination of organic solvent. This article reviews and summarizes the advantages, possible problems and solution and the feasibility of using microencapsulated genetic engineering cells in the treatment of cancer.
Animals ; Capsules ; Cell Transplantation ; Combined Modality Therapy ; Drug Compounding ; methods ; Genetic Engineering ; Genetic Therapy ; methods ; Mice ; Neoplasms, Experimental ; therapy ; Transfection

Objective To develop an effective and broad immune protective vaccination strategy by using DNA and recombinant vaccinia-based H5N1 vaccines.Methods BALB/c mice were immunized with various prime-boost regimens by using different DNA ( pIRES-based or pVRC-based) and recombinant vaccinia (Tiantan strain, rTTV)-based H5N1 vaccines expressing multivalent antigens (HA, NA, M1 and M2).The differences of immunity induced by two DNA vaccines were compared between intradermal electro -poration (IDE) and intramuscular electroporation (IME) deliveries.Immune responses were analyzed by hemagglutination inhibition( HAI) assay, neuraminidase ( NA)-specific antibody measured by ELISA , mi-croneutralization assay and IFN-γELISPOT assay .Results High levels of humoral immunity and T cell re -sponses were induced in mice primed with DNA-based vaccine than those primed with rTTVb-ased vaccine . DNA priming by IDE resulted in higher levels of neutralizing antibody in mice than those by IME delivery . Higher levels of HAI and anti-NA antibodies as well as NA-specific T cell responses were induced by pVRC-based DNA prime than those by pIRES-based DNA prime .HA-specific T cell responses were also enhanced in mice primed with pVRC-based DNA than those primed with pIRES-based DNA by IME .Conclusion The prime-boost strategies by using DNA-based vaccine in combination with rTTV-based H5N1 vaccine could induce humoral and T cell responses in mice against multi-antigens .Immunities induced by vaccines in com-bination might be modulated by various prime regimes .The study provided references for the further develop-ment of novel H5N1 vaccine and the optimization of immunization programs of combined multi-antigen vac-cine candidates .

Objective To construct a reverse genetic platform for influenza B virus and to rescue influenza B virus.Methods Eight plasmids carrying the gene segments of B/Florida/4/2006 virus were constructed by using the bidirectional promoter vector pHW2000.293T cells were co-cultured with MadinDarby canine kidney (MDCK) cells and then transfected with the eight plasmids.The supernatants of cell culture and cell debris were collected after transfection and then injected into embryonated chicken eggs and MDCK cells for rescuing the influenza B virus strains.Results This reverse genetic system could be used for the preparation of reassortant influenza B virus strains.The titers of hemagglutination units of the rescued virus achieved 128-256/50μl.Most of the reassortant virus particles were spherical under electron microscope.Conclusion The pHW2000 reverse genetic system could be used for the rescue of influenza B virus.Moreover,it could also be used for the construction of influenza B virus with specific mutations for further in vestigation on the characteristics of influenza B virus and the construction of vaccine strain.

Objective To investigate the possibility of using well-differentiated human airway epithelial cells (HAE) to isolate and identify human influenza A virus from a stale respiratory tract specimen.Methods The stale specimen used in this study was a nasopharyngeal swab specimen collected from a patient with unexplained pneumonia in Qinghai in 2010.It was positive for influenza A virus (H3N2) RNA, but negative for hemagglutination.Equal amount of the specimen was inoculated on HAE and on Madin-Darby canine kidney (MDCK) cells for virus isolation and passage.Cytopathic effects were observed daily after inoculation.Hemagglutination inhibition test was performed at every passage.Electron microscope was used to observe viral morphology.Viral genome was sequenced, followed by molecular evolutionary analysis.Results No progeny virus was isolated in MDCK cells, while a influenza A virus subtype H3N2 strain [A/Qinghai/178/2010(H3N2)] was isolated in HAE with a typical morphology and cytopathic effect of influenza A infection.The hemagglutination inhibition activity was 1∶16.Results of the molecular evolutionary analysis of viral genome showed that the influenza A virus (H3N2) strain was highly homologous to the A/Nanjing/1655/2010(H3N2) strain, which was isolated during the 2010 influenza pandemic in Nanjing.Conclusion HAE can be used for isolation and identification of virus from stale respiratory tract specimens.It is more sensitive than MDCK cells with regard to human influenza virus isolation.

Objective To quantitatively assess the virucidal activities of three commercial disin-fectants against human infected highly pathogenic avian influenza viruses subtype H5. Methods The 50%tissue culture infective dose ( TCID50 ) of avian influenza viruses was calculated. Quantitative suspension test was performed to evaluate the efficacy of three disinfectants. In that test, 105 TCID50 of avian influenza viru-ses were exposed to different disinfectants at different concentrations for different times with or without the in-terference with fetal bovine serum ( FBS) simulating the contaminated condition. The residual infectivity was determined by endpoint titration in Madin-Darby canine kidney ( MDCK) cells. The detail steps were that the mixture of viruses and disinfectants was inoculated at 37℃ with 5% CO2 for 1 hour. Then, it was re-placed by virus dilution medium and further incubated for 18 to 20 hours. ELISA was performed for the cal-culation of TCID50 . The titers of residual viruses were calculated according to Reed and Muench method. The low pathogenic avian influenza virus H9N2 was chosen as the control in this study. Results The re-mained infectivities of three viruses after 1 minute exposure to 1% Virkon solution were below the limit of de-tection (1. 0 lgTCID50/100 μl). Exposing to 0. 5% Virkon solution decreased the viral titers of H5N1 and H9N2 viruses below the detection limit and reduced the titer of H5N6 virus to 1. 75 lgTCID50/100 μl. The virucidal efficacy of 0. 25% Virkon solution against some of the detected viruses was achieved by increasing the exposure time to 5 minutes. The 84 Disinfectant solutions at concentrations of 10%, 5% and 2. 5% low-ered the viral titers of three viruses below the detection limit of 1. 0 lgTCID50/100 μl, but the 1. 25% 84 Disinfectant solution only lowered the viral titers to 1. 25-2. 5 lgTCID50/100 μl. The similar results were ob-served in groups treated with SOLARSEPT solutions. 1% 84 Disinfectant solution didn′t show any virucidal activity against the three viruses after 1 minute of exposure even when the exposure time was extended to 5 minutes. Under the contaminated condition, 1% Virkon solution, 10% and 5% 84 Disinfectant solutions as well as 100% and 50% SOLARSEPT solutions lowered the viral titers below 1. 0 lgTCID50/100μl. Conclu-sion The three commercial disinfectants (1% Virkon solution, 10% 84 Disinfectant solution and SOLAR-SEPT solution) were efficient virucides for highly pathogenic avian influenza viruses subtype H5 even under the contaminated condition. Increasing the exposure time had no significant effects on the efficacy of three disinfectants after the virucidal activities were neutralized by enough viruses. No significant differences in vi-rucidal activities of three disinfectants against HPAI H5 viruses and LPAI H9 virus were observed.

Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China.
Animals ; Antibodies, Viral ; immunology ; Electrophoresis, Polyacrylamide Gel ; Female ; Hemagglutination Inhibition Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; analysis ; immunology ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Rabbits ; Reassortant Viruses ; genetics ; immunology