环孢素和他克莫司在体外对乙型肝炎病毒复制影响的对比研究

Effects of ciclosporin and tacrolimus on rep lication of hepatitis B virus in vitro: a comparative study

目的 比较分析环孢素和他克莫司（FK506）在体外对乙型肝炎病毒（HBV）复制的影响。方法采用HBVDNA永久转染人肝癌细胞系HepG2．2．15细胞为体外实验模型，经四甲基偶氮唑蓝（MTT）试验确定环孢素及FK506对HepG2．2．15细胞生长无抑制作用的药物安全浓度后，将不同安全浓度的环孢素（0．6-20．0μg／ml）或FK506（5～100ng／ml）作用于该细胞系，每24小时换相应浓度新鲜含药培养基，药物作用4d后收集培养上清液和培养细胞待测。采用酶联免疫吸附试验检测上清液中乙型肝炎表面抗原（HBsAg）和乙型肝炎e抗原（HBeAg）水平，采用狭缝印迹杂交法半定量分析细胞内HBV DNA水平，综合评价环孢素和FK506对HBV病毒复制的影响。结果MTT结果显示环孢素的药物作用安全浓度为0～40．0μg／ml，FK506的安全浓度为0-400ng／ml。在安全浓度范围内，随着环孢素作用浓度的增加，其对HBsAg和HBeAg的抑制率逐渐上升，细胞内HBV DNA复制水平下降；当1．3、2．5及5．0μg／ml环孢素作用4d时，对HBsAg的抑制率分别为16．5％±9．4％、21．5％±8．9％和33．1％±5．3％，对HBeAg的抑制率分别为7．8％±2．2％、11．0％±2．3％和20．8％±1．5％，HBV DNA的表达量仅为对照组的56．1％±16．5％、41．7％±10．9％和39．5％±9．5％.在安全浓度范围内，FK506对HepG2．2．15细胞HBsAg和HBeAg的表达分泌，以及细胞内HBV DNA复制无明显抑制作用。结论环孢素在体外能呈剂量依赖性地抑制HBV复制；而FK506则无此作用。

Objective To investigate the effects of ciclosporin （CsA） and tacrolimus （FKS06） on replication of hepatitis B virus （HBV） in vitro. Methods HBV genome permanently transfected human liver cancer cells of the line HepG 2.2, 15 were cultured. CsA and FKS06 at different concentrations were added into the culture fluid so as to identify the nontoxic concentrations by MTT method. Then the HepG2. 2.15 cells were treated by CsA and FKS06 at different nontoxic concentrations respectively for 4 days. ELISA was used to detect the HB surface antigen （HBsAg） and HB e antigen （HBeAg） in the supematant. The relative replication level of HBV DNA was detected by slot blot analysis. Results MTT method confirmed that the nontoxic concentrations of CsA and FKS06 were 0-40. 0μg/ml and 0-400 ng/ml respectively. After the treatment of CsA at the concentration of 1.3, 2.5, and 5.0μg/ml, in comparison to the control group, the suppression rates of HBsAg expression in the HepG2.2. 15 cells were 16.5%±9.4% , 21.5%±8.9%, and 33.1%±5.3% respectively （ all P〈0.05 ） ; the suppression rates of HBeAg expression in the HepG2.2. 15 cells were 7. 8%±2.2%, 11.0%±2.3%, and 20. 8%±1. 5% respectively（all P〈0. 05）; and the HBV DNA replication levels were 56±16, 42±11, and 40±10 respectively （ P 〉 0.05, P 〈 0.05, and P 〉 0.05 ） . However, FKS06 at different nontoxic concentrations showed no significant inhibitory effect on the levels of HBsAg, HBeAg, and HBV DNA. Conclusion CsA dose- dependently inhibits the HBV replication in vitro, and FKS06 does not exercise similar effects.