RESUMO
Abstract Thymoquinone (TQ) has shown hepatoprotective effects in various experimental studies. We aimed to investigate the possible beneficial effects of TQ regarding its prevention of alpha-amanitin induced hepatotoxicity in human C3A hepatocytes. After administering alpha-amanitin in a concentrations of 1 and 10µg/mL on the cells in a hepatocyte cell line, TQ was administered in various concentrations (10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005 µg/mL). The MTT test was used to determine cell viability. For the groups given only TQ at various concentrations, the cell viability rates at 48 hours post-administration were found at 82.6, 98.3, 102.1, 102.5, 99.4, 99.4, 101.9 and 106.3%, respectively. For the group with 1μg/mL alpha-amanitin and various TQ concentrations, the cell viability rates were found at 74.6, 88.5, 87.4, 88.7, 85.7, 86.8, 88.4, and 92.9%, respectively. For the group with 10μg/mL alpha-amanitin and various TQ concentrations, the cell viability rates for each TQ subgroup were found at 65.2, 79.2, 81.4, 81.1, 81.8, 81.8, 82.2 and 91.9%, respectively. Our study is the first in vitro study that investigates TQ's effects on alpha-amanitin induced hepatotoxicity. Although TQ had beneficial effect in low doses did not significantly increase cell viability in liver damage due to alpha-amanitin toxicity.
Assuntos
Linhagem Celular/classificação , Técnicas In Vitro/métodos , Alfa-Amanitina/administração & dosagem , Fígado/fisiopatologiaRESUMO
Objective To construct a gene recombinant lentiviral vector pCMV -G -U6 -hHGF and detect its expression in C2C12 myoblast cells .Methods hHGF gene fragments were obtained and purified by RT -PCR method ,and were cloned to pCMV -G&NR -U6 ,then the restructured lentiviral vector was transformed into e . coli DH5 alpha ,the positive colonies were identified by BamHI and Hind Ⅲ enzyme digestion .The selected positive colonies were tested by PCR and sequencing analysis .The expression plasmids and packing plasmids were co -trans_fected into 293 T cells ,and virus titer was observed under the fluorescence microscope .Furthermore ,transfected C2C12 cells with lenti virus ,and the expression of HGF was detected by PCR and WB methods .ResuIts PCR and sequencing analysis showed that the lentiviral vector was constructed correctly and successfully ,the virus titer was above 1 x 109 IU/mL .The results of PCR and WB showed that HGF expression level in the lentiviral vector group was much higher than those of in blank control and negative control groups ,and yet the expression was stable after 72 hours .ConcIusion The lentiviral vector pCMV -G﹠NR -U6 -hHGF has been successfully constructed ,and stable expressed in C2C12 cells .It provides references for experimental study in the fields of the denervated skeletal muscle fibrosis and nerve regeneration treatment .
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Objective To study the hepatocyte cells infected by hepatitis C virus (HCV) positive serum. Methods Human hepatocyte 7701 was incubated with HCV RNA-positive and HCV antibody(Ab) negative sera BP52. Then, the expression of HCV antigen and the presence of HCV-RNA in cell and supernatant were assayed by RT-PCR, sequence analysis, immunofluorescent staining, Western blot, confocal laser microscopy. The ultrastructural changes of infected cells were observed by electro-microscopy. Results Plus-strand RNA and minus-strand RNA were intermittently detected in cell and/or supernatant on day 7-45 after infection. Sequence analysis demonstrated that the positive DNA nucleic acids were identified with HCV 5′-non-coding region(NCR) sequence. HCV core and NS3 protein were expressed in cytoplasm of infected cells. After 2 or 3 weeks, obvious intracellular ultrastructural changes and virus-like particles were observed. Conclusion human hepatocyte 7701 could support replication of HCV in vitro, which could be a useful tool for setting up cell model of HCV infection and studying the mechanism of HCV infection.