AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.1...AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.15 cells were treated with 2 pmol/L lamivudine for 16 d （lamivudine group）, cultured for 10 d, followed by 5 ng/ml TNF-α and 1000 U/mL IFN-γ, for 6 d （cytokine group）, or treated with 2 ~tmol/L lami- vudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ, for 6 d （sequential group）, or cultured without additions for 16 d （control group）. Intracellular DNA was extracted from 3 ×10^ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circu- lar DNA that may have remained. Both HBV covalently closed circular DNA （cccDNA） and HBV DNA were exam- ined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） were quantified with enzyme-linkedimmunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS： Compared to lamivudine alone （22.63%±0.12%）, both sequential （51.50% ± 0.17%, P = 0.034） and cytokine treatment （49.66% ± 0.06%, P = 0.041） showed a stronger inhibition of HBV cccDNA; the dif- ference between the.sequential and cytokine groups was not statistically significant （51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88）. The sequential group showed less inhibition of HBV DNA replication than the lamivudine group （67.47% ±0.02% vs 82.48% ± 0.05%, P = 0.014）; the difference between the sequen- tial and cytokine groups was not statistically significant （67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071）. The levels of HBsAg and HBeAg were significantly de- creased in the sequential treatment group compared to the other groups [HBsAg： 3.48 ± 0.04 （control）, 3.09 ± 0.08 （lamivudine）, 2.55± 0.13 （cytokine）, 2.32 ± 0.08 （sequential）, P = 0.042 for each between-group comparison; HBeAg 3.48 ± 0.01 （control）, 3.08 ± 0.08 （lamivudine）, 2.57 ± 0.15 （cytokine）, 2.34 ± 0.12 （se- quential）, P = 0.048 for each between-group compari- son]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells （58.03% ± 8.03% vs 100%, P = 0.000）. Lamivudine pretreat- ment significantly reduced IFN-γ, ± TNF-αmediated toxicity of HepG2.2.15 cells [85.82% =1= 5.43% （sequen- tial） vs 58.03% ± 8.03% （cytokine）, P = 0.002]. CONCLUSION： Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-y and TNF-α by decreasing the viral load.展开更多
基金Supported by Zhongshan Youth Foundation of Fudan University,China,No.257
文摘AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.15 cells were treated with 2 pmol/L lamivudine for 16 d （lamivudine group）, cultured for 10 d, followed by 5 ng/ml TNF-α and 1000 U/mL IFN-γ, for 6 d （cytokine group）, or treated with 2 ~tmol/L lami- vudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ, for 6 d （sequential group）, or cultured without additions for 16 d （control group）. Intracellular DNA was extracted from 3 ×10^ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circu- lar DNA that may have remained. Both HBV covalently closed circular DNA （cccDNA） and HBV DNA were exam- ined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） were quantified with enzyme-linkedimmunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS： Compared to lamivudine alone （22.63%±0.12%）, both sequential （51.50% ± 0.17%, P = 0.034） and cytokine treatment （49.66% ± 0.06%, P = 0.041） showed a stronger inhibition of HBV cccDNA; the dif- ference between the.sequential and cytokine groups was not statistically significant （51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88）. The sequential group showed less inhibition of HBV DNA replication than the lamivudine group （67.47% ±0.02% vs 82.48% ± 0.05%, P = 0.014）; the difference between the sequen- tial and cytokine groups was not statistically significant （67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071）. The levels of HBsAg and HBeAg were significantly de- creased in the sequential treatment group compared to the other groups [HBsAg： 3.48 ± 0.04 （control）, 3.09 ± 0.08 （lamivudine）, 2.55± 0.13 （cytokine）, 2.32 ± 0.08 （sequential）, P = 0.042 for each between-group comparison; HBeAg 3.48 ± 0.01 （control）, 3.08 ± 0.08 （lamivudine）, 2.57 ± 0.15 （cytokine）, 2.34 ± 0.12 （se- quential）, P = 0.048 for each between-group compari- son]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells （58.03% ± 8.03% vs 100%, P = 0.000）. Lamivudine pretreat- ment significantly reduced IFN-γ, ± TNF-αmediated toxicity of HepG2.2.15 cells [85.82% =1= 5.43% （sequen- tial） vs 58.03% ± 8.03% （cytokine）, P = 0.002]. CONCLUSION： Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-y and TNF-α by decreasing the viral load.
