拉米夫定单独和与α干扰素序贯处理的人肝胚瘤细胞株HepG2.2.15HBV差异复制的研究

目的 研究LAM单独和与IFN-序贯处理对人肝胚瘤细胞株HepG2.2.15细胞的HBV复制的影响,探讨LAM与IFN-α在体外抑制HBV复制的差异.方法 以未处理的HepG2.2.15细胞作为对照组;以1 000 IU/ml浓度IFN-α连续处理HepG2.2.15细胞10 d为单独IFN-α处理组;以0.2、1、5、20、100 μmol/L的LAM处理HepG2.2.15细胞为单独LAM处理组;序贯处理组则以浓度为0.04、0.2、5、25、125、200μmoL/L的LAM连续处理HepG2.2.15细胞7 d,再补充1 000 IU/ml IFN-α与LAM联合处理3 d,然后停止LAM处理,再单独以1 000 IU/ml IFN-α连续处理细胞10 d;分别用ELISA法、点杂交和Southern杂交分析不同处理时期、不同处理组HepG2.2.15细胞分泌的HBV抗原、细胞外HBV DNA、细胞内HBV复制中间体DNA以判断HepG2.2.15细胞内HBV复制情况.结果 LAM连续处理至10 d时,单独LAM处理组HepG2.2.15细胞分泌的HBsAg分别是1.77±0.22、1.65±0.25、1.95±0.19、1.34±0.11、1.07±0.05,分泌的HBeAg是1.41±0.13、1.37±0.09、1.63±0.07、1.26±0.12、1.05 ±0.09,对照组分泌的HBsAg和HBeAg分别是3.34±0.15和3.33±0.05,单独LAM处理组与对照组相比分泌的HBsAg和HBeAg下降,差异有统计学意义(HBsAg的t值为10.21、10.04、9.94、18.62、24.86,HBeAg的t值为23.87、32.97、34.22、27.57和38.35,P均＜0.05).点杂交、Southern杂交分析显示LAM连续处理10 d后,单独LAM处理组的细胞外HBV DNA和细胞内HBV复制中间体DNA不能被检测到.停止LAM处理并代之以1 000 IU/ml IFN-α序贯处理10 d,序贯处理组均有细胞内HBV复制中间体DNA的出现,且细胞外HBV抗原和HBV DNA恢复表达;即HBV颗粒在HepG2.2.15细胞内又重新恢复复制状态并分泌至细胞外.结论 LAM与IFN-α在体外细胞模型中具有不同的抗病毒效应,导致HepG2.2.15细胞内HBV复制的差异性.

Abstract：

Objective To investigate the characteristics of HBV replication in HepG2.2. 15 cells treated with LAM alone or sequentially treated with LAM and IFN-α, and to further explore the different suppressive effect on HBV replication by LAM and IFN-α in vitro. Methods Untreated HepG2. 2. 15 cells were used as control group, HepG2. 2. 15 cells treated with 1 000 IU/ml of IFN-α alone for 10 d were served as IFN-α group. The HepG2.2. 15 cells treated with 0.2,1,5,20,100 μmol/L of LAM were used as LAM groups. HepG2. 2. 15 cells treated with 0. 04,0. 2,5,25,125,200 μmol/L of LAM for 7 d, then combined with 1 000 IU/ml of IFN-α for another 3 d. Afterwards, the cells were treated with 1 000 IU/ml of IFN-α only for another 10 d. These cells were served as LAM/IFN-α sequential treatment group. The ELISA was used for analyzing the secreted HBV antigens, while the Dot blot, Southern blot were applied for analyzing the extracellular HBV DNA and intracellular HBV replicative intermediate DNA in HepG2. 2. 15 cells of different treatment groups. Results The secreted HBsAg in the LAM group were 1. 77 ± 0. 22, 1.65 ±0.25, 1.95 ±0. 19, 1.34 ±0. 11, 1.07 ±0.05, respectively, and the secretion of HBeAg were 1.41 ±0. 13, 1.37 ± 0. 09, 1.63 ± 0. 07, 1.26 ± 0. 12, 1.05 ± 0. 09. The secreted HBsAg and HBeAg in control group were 3. 34 ± 0. 15 and 3.33 ± 0. 05. Statistical analysis showed that HBsAg and HBeAg secretion in the LAM group were significantly reduced by treatment with LAM. The t values of HBsAg were 10. 21,10.04, 9.94, 18.62, 24.86, and the t values of HBeAg were 23.87, 32.97, 34.22, 27.57, 38.35,respectively, all P values were ＜ 0.05. Dot blot, Northern blot hybridization analysis indicated that the extracellular HBV DNA and intracellular HBV replicative intermediate DNA could not be detected in the LAM group after cells treated by LAM for 10 days. When LAM was replaced with treatment of 1 000 IU/ml of IFN-α alone, it could not suppress the HBV replication effectively. Moreover, the intracellular HBV replicative intermediate DNA still existed in almost all groups, accompanied with the recovered expression of HBV antigens as well as extracellular HBV DNA, which suggested that the HBV particles restored replication again and secreted extracellular in HepG2. 2. 15 cells, although the sequential treatment lasted for 10 days.Conclusion The effect of viral suppression by LAM and IFN-α in vitro were different, which attributed to the different HBV replicative characteristcs in HepG2. 2. 15 cells.