| Thymosin α1 (Tα1) was discovered in 1984 which was secreted fromthymus, it was an important T-lymphocyte regulatory factor, concerned withcellular immunity. Recently it has been extensively applied to treat all kinds ofimmunodeficiency disease, auto-immune disease, tumor, virus and othermicroorganism infection. As a biological response modifiers(BRMs), Tα1could recover the function of cytotoxic T-lymphocyte and natural killer whichplay an important role in antivirus, promote the production of the IL-2 and othercytokine and its receptor. Thus it could enhance the function of organismantiviral immunity and clear internal virus favorably. At present therapeuticeffect of viral hepatitis B is not satisfactory, as a new immunological modifiers,Tα1has aroused the extensive attention of domestic and foreign researchers.Objective:For further explorate and utilize the drugs of anti HBV and influenza virus,in this study, the pharmacodynamic activity of Tα1were evaluated and wereexplorated its mechanism of antivirus through antivirus experiment in vitro andvivo.Methods:1.cultivation of 2.2.15 cellAccording to routine method to resuscitate 2.2.15 cell. As 2.2.15 celloverflowed full culture flask, digest with diastasevera and centrifuged cell,1:3 passage, about 7 day overflowed full culture flask . conserve one part ofcell long-term in -80℃ that had been added to freeze-preserve fluid, continueto cultive another part of cell which culture solution , so that we could dofollowing experiment.2.detection of HBV markers in 2.2.15 cellDuring the cultivation of 2.2.15 cells, supernatations were collectedrespectively in 3d, 5d, 7d, 10d, 14d and preserved at -20℃. Hepatitis B surfaceantigen(HBsAg) and Hepatitis B e antigen (HBeAg) were detected by ELISA.HBV-DNA were examined by real-time quantantitative PCR.3.experiment of drugs toxicityDifferent concentration of Tα1were added to 2.2.15 cells, then exchanged thedrugs every 3 days until to 8d, observed cytopathic effect(CPE) under inversionmicroscope in order to evaluate the toxicity of Tα1to 2.2.15 cells.4.anti-HBV experiments in vitroDifferent concentration of Tα1and 3TC were added to 2.2.15 cells, thenexchanged the drugs every 3 days until to 14d. After treated with drugs fordifferent times, the culture fluids are collected and the level of HBsAg andHBeAg were detected using Enzyme Linked Immunosorbent Assay(ELISA),then antigen inhibitory rate and IC50 were calculated. The copies of HBV-DNAwere measured by real-time quantantitative PCR.5.anti-influenza virus experiments in vivoIn vivo, after intranasal infection with 15LD50 influenza A virus, the impactswhich the various doses of Tα1 on lung index of mice infected withinfluenza A virus were observed, Ribavirin was used as a positive control.Results:1.detection of HBV markers in 2.2.15 cellsIn this study, on the third day of cultivation, HBsAg, HBeAg and HBV-DNAcould be detected, their quantities increased continuesly until the 14th day.2.results of cytotoxicity experiments of Tα1The experiment results of cytopathic effect(CPE) show that the TC50 ofTα1 are 131.3μg/ml, the maximum nontoxic concentration(TC0) are 78μg/ml.3.The results of antivirus experiment show that in concentration of9.75μg/ml ～ 78μg/ml, the inhibitory rates of Tα1 on HBsAg , HBeAggradually enhanced with the concentration of Tα 1 increasing (HBsAg:IC50=36.6μg/ml, TI=3.58), (HBeAg: IC50=29.93μg/ml, TI=4.39)respectively(P<0.01);The copies of DNA decreased obvious with theconcentration of Tα1 increasing, it show that Tα1 had inhibitory activity onHBV-DNA obviously.4.anti-influenza virus experiments in vivoThe results of animals experiment show that the mice lung index in the dosesof 0.8mg/kg, 0.4mg/kg, 0.2mg/kg are 1.15, 1.21, 1.29 respectively;inhibitory ratesof lung index are 17.41%,13.10 %,7.66% respectively, Tα1could reduce themice lung index and extenuation the lung pathological changes obviously.Conclusions:1.2.2.15 cells cultivated in vitro could express HBsAg and HBeAg stabily,and the level of HBV-DNA was stable. It suggested that 2.2.15 cells be used ascell model to screen anti-HBV drugs.2.The results of anti-HBV experiment in vitro show that the maximumnontoxic concentration(TC0)Tα1 was 78μg/ml. Tα1within 9.75μg/ml and 78μg/ml had obviously inhibitory activity on HBsAg, HBeAg and HBV-DNA.The inhibitory rates gradually increased with the concentration of Tα 1increasing and reflect certain dose-effect relationship. The results suggest thatTα1 had inhibitory activity on HBV in vitro and the value of further study inclinic.3.Experiment effects anti-influenza in vivo, Tα1at the doses of 0.2mg/kg~0.8mg/kg could decrease the pneumonia symptom of mice infectd by influenza Avirus, high dosage Tα1 had significantly inhibitory activity on influenza Avirus, compared with virus control, the difference is significant(P<0.01). Theresults indicate that Tα1 has inhibitory activity on influenza, and it shows itsgood value of further study and utilization in clinic.
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