细胞毒性化疗药物表柔比星对HBV复制水平影响的体外试验

Effect of cytotoxic chemotherapy drug epirubicin on hepatitis B virus replication in vitro

目的:在体外研究细胞毒性化疗药物表柔比星对乙肝病毒(hepatitis B virus,HBV)复制水平的影响。方法:3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法检测0、0.1、0.5、1.0μmol/L不同浓度表柔比星对HBV稳定细胞系Hep G2.2.15细胞和Hep G2-HBV1.1细胞活力的影响。将药物直接添加在Hep G2.2.15细胞和Hep G2-HBV1.1细胞中,提取细胞内及上清中的HBV复制中间体,通过real-time PCR法和southern-blot法检测HBV DNA拷贝量。结果:Hep G2.2.15细胞分别经0、0.1、0.2、0.5μmol/L表柔比星处理后,细胞内HBV病毒拷贝量(×106)分别是3.53±0.26、5.39±0.23、9.40±0.92、19.07±1.47,各组间有统计学差异(F=10.984,P=0.005)。同样,Hep G2-HBV1.1细胞也分别经0、0.1、0.2、0.5μmol/L表柔比星处理后,细胞内HBV病毒拷贝量(×106)分别是2.80±0.37、5.14±0.49、14.24±1.29、26.59±1.98,各组间有统计学差异(F=13.122,P=0.003)。Southern-blot检测表明Hep G2.2.15细胞内HBV DNA复制中间体相对量在对照组(0μmol/L表柔比星)和处理组(0.5μmol/L表柔比星)中分别为(1.00±0.01)和(1.53±0.05),对照组和药物处理组比较有统计学差异(t=19.202,P=0.002)。同样,Hep G2-HBV1.1细胞内HBV DNA复制中间体相对量,在对照组和处理组中分别为1.01±0.02、3.12±0.20,对照组和药物处理组比较有统计学差异(t=18.034,P=0.003)。上述结果提示表柔比星促进细胞内HBV的复制。Hep G2.2.15细胞分别经0、0.1、0.2、0.5μmol/L表柔比星处理后,细胞上清液中HBV病毒拷贝量(×106)分别是2.37±0.22、3.50±0.27、6.02±0.56、17.74±0.96,各组间有统计学差异(F=20.726,P=0.001)。同样,Hep G2-HBV1.1细胞也分别经0、0.1、0.2、0.5μmol/L表柔比星处理后,细胞上清液中HBV病毒拷贝量(×106)分别是3.75±0.35、6.05±0.48、10.28±1.01、20.86±1.81,各组间有统计学差异(F=14.761,P=0.002)。Southern-blot检测表明Hep G2.2.15细胞上清中HBV DNA复制中间体相对量,在对照组和药物处理组中分别为1.08±0.02和2.69±0.05,对照组和药物处理组比较有统计学差异(t=58.33,P=0.000)。同样,Hep G2-HBV1.1细胞内HBV DNA复制中间体相对量,在对照组和药物处理组中分别为1.03±0.01、3.52±0.05,对照组和药物处理组比较有统计学差异(t=85.801,P=0.000)。该结果提示表柔比星促进细胞分泌或释放更多的HBV到细胞上清液中。结论:表柔比星有直接的促HBV复制的作用,该作用可能成为化疗后HBV再激活的新机制。

Objective ：To study the effect of cytotoxic chemotherapy drug epirubicin on hepatitis B virus（HBV） replication in vitro. Methods：The cell viability after epirubicin treatment was detected by MTT assay. The epirubicin was added into the culture of two stable HBV-expression cell lines（HepG2.2.15 cells and HepG2-HBVI.1 cells）. The HBV replication intermediates were extracted from the cytoplasma and supernatant,and then HBV DNA copies were detected by quantitative fluorescent PCR and Southern blot. Results：HepG2.2.15 cells were treated by 0,0.1, 0.2,0.5 μmol/L epirubicin respectively, and the intracellular HBV DNA copies（ x 106） were 3.53 ± 0.26,5.39± 0.23,9.40 ±0.92,19.07 ＋ 1.47;there were significant differences among these groups（F=10.984,P=0.005）. Similarly, HepG2-HBVI.1 cells were treated by 0,0.1,0.2,0.5 μmol/L epirubicin respectively,and the intracellular HBV DNA copies（×10^6） were 2.80± 0.37,5.14 ± 0.49, 14.24± 1.29,26.59 ±1.98;there were significant differences among these groups （F=13.122, P=-0.003）. The Southern blot showed that the relative volume of HBV DNA replicative intermediate in HepG2.2.15 cells were 1.00 ±0.01 and 1.53 ± 0.05 in control group and drug group respectively;there were significant differences between two groups （t=19.202 ,P=-0.002）. Similarly, the relative volume of HBV DNA replicative intermediate in HepG2-HBVI.1 cells were 1.01 ± 0.02 and 3.12 ±0.20, there were significant differences between two groups （t = 18.034, P=0.003 ）. These results suggested that epirubicin promoted intracellular replication of HBV. Meanwhile, HepG2.2.15 cells were treated by 0,0.1,0.2,0.5 p, mol/L epirubicin respectively, and the HBV DNA copies （ × 10^6） in the supernatant were 2.37 ± 0.22,3.50 ± 0.27,6.02 ± 0.56,17.74 ± 0.96,there were significant differences among these groups（F=20.726, P=-0.001 ）. Similarly, HepG2-HBV 1.1 cells were treated by 0,0.1,0.2,0.5μmol/L epirubicin respectively, and the HBV DNA copies （×10^6） in the supernatant were 3.75 ± 0.35,6.05 ± 0.48,10.28 ± 1.01,20.86 ± 1.81, there were significant differences among these groups （F=14.761 ,P=0.002）. The Southern blot showed that the relative amount of HBV DNA replicative intermediate in the supernatant of HepG2.2.15 cells were 1.08 ± 0.02 and 2.69±0.05 in control group and drug group respectively; there were significant differences between two groups （t=58.33,P=0.000）. Similarly, the relative amount of HBV D NA replicative intermediate in the supernatant of HepG2-HBVI.1 cells were 1.03 ± 0.01 and 3.52 ± 0.05 in control group and drug group respectively ,there were significant differences between two groups（t=85.801 ,P=0.000）. The results indicated that epirubicin promoted cells secreting or releasing HBV particles. Condusion：Epirubicin has a direct role in promoting HBV replication,which may be a new mechanism of HBV reactivation after chemotherapy.