Objective To investigate the effect of TLCK(N-alpha-tosyl-L-lysyl-chloromethylketone) on acute hemorrhagic necrotizing pancreatitis(AHNP) associated with lung injury.Methods Seventy SD rats were randomized into seven groups(n=10 in each group): group1,normal control group(rats were treated with 0.9% sodium chloride i.v.);group 2,AHNP control group(AHNP rats were treated with 0.9% sodium chloride i.v.);group 3,AHNP rats were treated with 5?g/kg TLCK;group 4,AHNP rats were treated with 10?g/kg TLCK;group5,AHNP rats were treated with 20 ?g/kg TLCK;Group3,4,5were treated with TLCK i.v.immediately after the establishment of SAP rat model;group 6,AHNP rats were treated with TLCK i.v.30 minutes before the establishment of AHNP model;group 7,AHNP rats were treated with TLCK i.v.30 minutes after the establishment of AHNP rat model;Group 6,7 were treated with 10 ?g/kg TLCK.Group 1-7 were used to select the best dosage and administration time of TLCK by observing the survival rate for seven days.Then,according to the result of selecting,the SD rats were divided into three groups(n=6 in each group): group N,Sham operated control group(0.9% sodium chloride was reversely injected into SD rat′s pancreas and rats were treated with 0.9% sodium chloride i.v.);group P: AHNP group;group T: TLCK treatment group(AHNP rats were treated with 10 ?g/kg TLCK immediately after the establishment of AHNP rat model).The rats of the last three groups were killed 6 hours after operation.Alveolar macrophages(AMs) were harvested by bronchoalveolar lavage.The protein content of bronchoalveolar lavage fluids(BALF),the myeloperoxidase(MPO) of lung tissue,and tumor necrosis factor ?(TNF?) secreted by AMs were determined.The expression of NF-?B in AM was checked as well as lung histology.Results The survival rate of group 1-7 was100%,0%,70%,100%,80%,0%,and 90% respectively.The level of MPO of lung tissue and protein content of BALF ingroup N were lower than that,in groups P and T(P

Objective To investigate the effects of application of pseudoreplica technique in electron microscope observation of A?.Methods With the modification of classical pseudoreplica technique,ADDLs were spotted and concentrated on the gel before negative staining.Results The structures of 5nm to10nm spherical granules and 20 to 30nm coils were visualized by transmission electron microscopy(TEM).Conclusion Pseudoreplica is rapid and effective in ADDLs condensation,negative staining especially in further ultra miero structure observation and research.

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Objective To investigate whether the apoptosis-2 ligand (Apo-2L),known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),could enhance irradiation-induced apoptosis in lung adenocarcinima H1975 cells that are resistant to the epithelial growth factor receptor (EGFR)-TKI.Methods Adenocarcinima H1975 cells were randomly divided into four groups:the control group,Apo2L group,irradiation group,and both Apo-2L and irradiation group.H1975 cells were pretreated with Apo2L under different concentrations of 200 and 228 ng/ml at 24 h before irradiation with doses of 1,1.5,2,2.5,3,3.5 and 4 Gy.The apoptosis rates of all groups were analyzed by flow cytometry 24 h post-irradiation.The inhibition rates of cell proliferation were measured by the MTT assay.Results MTT assay showed that the Apo-2L treatment significantly inhibited cell proliferation(x2 =136.17,P ＜ 0.05).The apoptosis rates of the four groups were different significantly,and the apoptosis rate of radiation combined with drug group was significantly higher than the other three groups(x2 =78.02,P ＜0.05).Conclusions The Apo-2L could not only inhibit the proliferation but also promote radiation-induced apoptosis of adenocarcinoma H1975 cells.

To develop a recombinant lentivirus co-expressing structural protein of hepatitis C virus (HCV) and secreted Gaussia Luciferase (Gluc), we first constructed an expression vector that encoded HCV structural protein (C, E1, E2) and GLuc named pCSGluc2aCE1E2. The expression of HCV proteins and Gluc was confirmed by an immunofluorescence assay (IFA) and the detection of luciferase activity. Recombinant lentivirus (VSVpp-HCV) was developed by the co-transfection of pCSGluc2aCE1E2 into 293T cells with pHR'CMVA8.2 and pVSVG. The infectivity of VSVpp-HCV was confirmed by luciferase activity detection, IFA and western blotting. Virus-like particles were identified using electron microscopy after concentration. The results showed that the level of luciferase activity correlated with the expression of HCV protein after the infection of cells with lentivirus VSVpp-HCV. Therefore, the expression level of HCV proteins could be evaluated by detecting the luciferase activity of Gluc. In conclusion, this research pave a way for the development of transgenic mice that express HCV proteins and Gluc, which enable the evaluation of anti-HCV therapy and vaccine in vivo.
Animals ; Copepoda ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Hepacivirus ; genetics ; metabolism ; Hepatitis C ; virology ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

Interactions between noroviruses (NoVs) and the receptors of histo-blood group antigens (HB-GAs) affect the infectivity and host susceptibility of NoVs. We elucidated the binding profile of a GII. 12 NoV to HBGAs. First, we synthesized the P domain sequence of the GII. 12 NoV strain Pune (GenBank accession number EU921353). Protein of the P domain was expressed in a prokaryotic system. Formation of the P particle was monitored by gel-filtration chromatography. Antiserum was prepared by immunization of mice with GII. 12 P particles. The binding profile of the GII. 12 NoV Pune strain was determined by binding of the P particle with a panel of saliva samples with various known HBGAs phenotypes. The GII. 12 NoV was bound strongly to saliva samples of subjects with B and AB types and weakly to A, O secretor, and non-secretor saliva samples, suggesting higher affinity with B antigen by GII. 12 NoV. These results were consistent with those determined by a previous crystallography study of GII. 12 NoV. These data suggested that individuals with B and AB blood types may be more susceptible to infection by GII. 12 NoV compared with those with other blood types. Our findings may provide a basis for the prevention and control of an epidemic of GII. 12 NoV.
Animals ; Blood Group Antigens ; metabolism ; Caliciviridae Infections ; metabolism ; virology ; Female ; Gastroenteritis ; metabolism ; virology ; Genotype ; Humans ; Mice ; Mice, Inbred BALB C ; Norovirus ; genetics ; metabolism ; Protein Binding ; Receptors, Virus ; metabolism ; Viral Proteins ; genetics ; metabolism

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.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is a new member in the genus Phlebovirus of the family Bunyaviridae identified in China. The SFTSV is also the causative pathogen of an emerging infectious disease: severe fever with thrombocytopenia syndrome. Using immunofluorescent staining and confocal microscopy, the intracellular distribution of nucleocapsid protein (NP) in SFTSV-infected THP-1 cells was investigated with serial doses of SFTSV at different times after infection. Transmission electron microscopy was used to observe the ultrafine intracellular structure of SFTSV-infected THP-1 cells at different times after infection. SFTSV NP could form intracellular inclusion bodies in infected THP-1 cells. The association between NP-formed inclusion bodies and virus production was analyzed: the size of the inclusion body formed 3 days after infection was correlated with the viral load in supernatants collected 7 days after infection. These findings suggest that the inclusion bodies formed in SFTSV-infected THP-1 cells could be where the SFTSV uses host-cell proteins and intracellular organelles to produce new viral particles.
Cell Line ; China ; Humans ; Inclusion Bodies, Viral ; ultrastructure ; virology ; Macrophages ; ultrastructure ; virology ; Phlebotomus Fever ; virology ; Phlebovirus ; genetics ; physiology ; ultrastructure ; Thrombocytopenia ; virology

Objective To express and characterize the virus-like particles( VLPs) of H5 subtype containing of hemagglutinin ( HA ) and matrix 1 ( M1 ) protein by using Baculovirus-insect cells .Methods Full length genes encoding HA protein from the A/Indonesia/05/2005(H5N1) strain and the M1 protein from the A/Anhui/01/2005 ( H5N1 ) strain were cloned into a baculovirus expression vector to construct pFBD-M1-HA.The expression of HA and M1 proteins were detected by Western blot and indirect immunoflu-orescence after the transfection of Spodoptra frugiperda (Sf9) insect cells with recombinant baculovirus.Pu-rified VLPs were analyzed by SDS-PAGE and visualized with transmission electron microscope.The biologi-cal activity of purified VLPs was detected by hemagglutination test.Results The HA and M1 proteins of H5 subtype expressed by baculovirus-insect cells could be self-assembled into the functional mature VLPs.The hemagglutination titer of VLPs was as high as 1024 HAU/50μl.Conclusion The H5 subtype VLPs as pre-pared in this study would pave a way for the development of a candidate recombinant A ( H5) vaccine.