| Thymosinα1 (Tα1) is a kind of peptide hormone which was secreted from thymus,it has the function of immunological regulation,anti-virus,anti-tumor.Recently it has been extensively applied to treat all kinds of immunodeficiency disease, auto-immune disease, tumor,virus and othermicroorganism infection. At present therapeutic effect of viral hepatitis B is not satisfactory,as a new immunological modifiers,Tα1has aroused the extensive attention of domestic and foreign researchers.Object:In our study,the pharmacodynamic activity of Tα1were evaluated and were explorated its mechanism of antivirus through antivirus experiment in vitro of anti-HB virus and vivo of anti-Influenza virus,to research the mechanism of Tα1 and for the further explorate and utilize the drugs of anti HBV and influenza virus.Method:(1)cultivation of HepG2.2.15 cellAccording to routine method to resuscitate HepG2.2.15 cell. As the cell overflowing full culture flask, digest with diastasevera and centrifuged cell,1:3 passage, about 3-5 days overflowed full culture flask . conserve one part of cell long-term in -80℃that had been added to freeze-preserve fluid, continue to cultive another part of cell which culture solution ,to continue the following experiment.(2)detection of HBV markersWe make the Hepatitis B surface ntigen(HBsAg) and Hepatitis B e antigen (HBeAg) as the markers.The passage cell of HepG2.2.15 were curtured at 37℃,5%CO2 ,for a day then change the nutrient solution,then the supernatants were collected espectively in 3d, 5d, 7d, 10d, 14d and preserved at -20℃. HBsAg and HBeAg were detected by ELISA. BV-DNA and were examined by real-time quantantitative PCR.(3)experiment of Tα1 toxicityDifferent concentration of Tα1were added to 2.2.15 cells, after 8d of culture , observed cytopathic effect(CPE) under inversion microscope in order to evaluate the toxicity of Tα1to 2.2.15 cells.(4)anti-HBV experiments in vitroDifferent four concentrations that below Tc50 of Tα1were added to 2.2.15 cells,then exchanged the drugs every 3 days until to 9d.The supernatant fluids are collected and the level of HBsAg and HBeAg were detected using ELISA,then antigen inhibitory rate and IC50 were calculated. The copies of HBV-DNA were measured by real-time quantantitative PCR.(5)anti-influenza virus experiments in vivo Ribavirin was used as a positive control,administration 15LD50 influenza A virus, the impacts which the various doses of Tα1 on lung index of mice infected with influenza A virus were observed.Result:(1)detection of HBV markers in 2.2.15 cells In this study,on the third day of cultivation, HBsAg, HBeAg and HBV-DNA could be detected, and their quantities increased continuesly until the 11th day.(2)results of cytotoxicity experiments of Tα1 The experiment results of cytopathic effect(CPE) show that the TC50 of Tα1 are 229.6μg/ml, the maximum nontoxic concentration(TC0) are 100μg/ml.(3)Results of anti-HBV experiments in vitro The result shows that in concentration of 20μg/ml-80μg/ml,Tα1 have the inhibitory effect on HbsAg and HBesAg.Inhibitory rate gradually enhanced with the concentration of Tα1 increasing.and the inhibitory rates are 48.5%-56.79%,47.04%-55.31% on HbsAg and HbesAg.And the effect on HbsAg is stronger.The IC50 of these two virus are 24.42,29.61μg/ml,therapeutic index are 9.4,7.75;The copies of DNA in supernatant decreased obvious with the concentration of Tα1 increasing showing that Tα1 had inhibitory activity on HBV-DNA obviously,inhibitate rate are being 31.73%-68.07%.(4)Result of anti-influenza virus experiments in vivoTα1of 0.4mg/kg,0.8mg/kg doses could reduce the mice lung index and extenuation the lung pathological changes obviously contrast with the model,and the difference between them was significant(P<0.01).Conclusions:(1)HepG2.2.15 cells cultivated in vitro could express HBsAg and HBeAg stabily,and the level of HBV-DNA was stable suggesting that HepG2.2.15 cells be used as cell ideal model to screen anti-HBV drugs.(2)Tαbetween 20μg/ml and 80μg/ml has obviously inhibitory activity on HBsAg, HBeAg and HBV-DNA.The inhibitory rates gradually increased with the concentration of Tα1increasing and reflect certain dose-effect relationship that results suggest that Tα1 has the value of further study in clinic. (3)Anti-influenza Experiment in vivo of mice manifest that Tα1at the doses of 0.2mg/kg-0.8mg/kg could both decrease the pneumonia symptom of mice infectd by influenza virus, high dosage Tα1 had significantly inhibitory activity on influenza virus , and the 0.4mg/kg,0.8mg/kg have significant difference(P<0.01) comparing with model indicating that Tα1 has inhibitory activity on influenza virus.
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