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  

Study of the different HBV replication in HepG2. 2. 15 cells treated with lamivudine or sequentially treated with lamivudine and interferon-α

作　　者：管世鹤[1] 杨凯[1] 卢银平[2] 杨东亮[3] 

机构地区：[1]安徽医科大学第二附属医院检验科 ,合肥230601 [2]华中科技大学同济医学院附属协和医院病毒研究室 [3]华中科技大学同济医学院附属同济医院临床免疫研究室

出　　处：《中华检验医学杂志》2011年第3期265-270,共6页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（30600522）

摘　　要：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1000IU／ml浓度IFN—α连续处理HepG2．2．15细胞10d为单独IFN—α处理组；以0．2、1、5、20、100mol／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0．0d、0．2、5、25、125、200Ixmol／L的LAM连续处理HepG2．2．15细胞7d，再补充1000IU／mlIFN—α与LAM联合处理3d，然后停止LAM处理，再单独以1000lU／mlIFN—α连续处理细胞10d；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HBVDNA、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10d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、10．0d、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10d后，单独LAM处理组的细胞外HBVDNA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1000Iu／mlIFN—α序贯处理10d，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HBVDNA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。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 〈 O. 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 intracellul

关 键 词：细胞系 肿瘤 肝炎病毒 乙型 病毒复制 拉米夫定 干扰素Α 

分 类 号：R512.62[医药卫生—内科学]