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

发布时间：2019-04-09 07:19

【摘要】：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。
[Abstract]:Aim: to study the effect of ecrobicin on the replication of hepatitis B virus (hepatitis B virus,HBV) in vitro. Methods: 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide, 5-(4,5-dimethylthiazolyl)-2,5-diphenylthiazolyl-2,5-diphenyltetrazolium The effects of 0, 0.1, 0.5 and 1.0 渭 mol / L Epiromycin on the viability of HBV stable cell line Hep G2.2.15 and Hep G2-HBV1.1 cells were detected by MTT] assay. The drug was added directly to Hep G2.2.15 cells and Hep G2-HBV1.1 cells to extract the HBV replication intermediates in the cells and supernatant. The amount of HBV DNA replication was detected by real-time PCR and southern-blot methods. Results: after Hep G2.2.15 cells were treated with 0, 0.1, 0.2 and 0.5 渭 mol / L Epiromycin respectively, the intracellular HBV virus copy amount (脳 10 6) was 3.53 卤0.26,5.39 卤0.23,9.40 卤0.92, 19.07 卤1.47,respectively. There were statistical differences among the three groups (F = 10.984, P < 0.005). Similarly, Hep G2-HBV1.1 cells were treated with 0, 0.1, 0.2 and 0.5 渭 mol / L Epiromycin, respectively, and the intracellular HBV virus (脳 10 6) was 2.80 卤0.37, 5.14 卤0.49, 14.24 卤1.29,26.59 卤1.98, respectively. There were statistical differences among the three groups (F = 13.122, P < 0.01). The relative amount of HBV DNA replication intermediates in Hep G2.2.15 cells was (1.00 卤0.01) in the control group (0 渭 mol / L Epiromycin) and in the treatment group (0.5 渭 mol / L Epiromycin), respectively. The relative amount of HBV DNA replication intermediates in Hep G2.2.15 cells was (1.00 卤0.01). And (1.53 卤0.05), There was a significant difference between the control group and the drug treatment group (t = 19.202, P = 0.002). Similarly, the relative amount of HBV DNA replication intermediates in Hep G2-HBV1.1 cells was 1.01 卤0.02 in the control group and 3.12 卤0.20 in the treatment group, and there was a significant difference between the control group and the drug treatment group (t = 18.034, P < 0.003). The above results suggested that Epiromycin promoted the replication of intracellular HBV. Hep G2.2.15 cells were treated with 0, 0.1, 0.2 and 0.5 渭 mol / L Epiromycin, respectively, and the cells were treated with 0, 0.1, 0.2 and 0.5 渭 mol / L EPROX, respectively. The amount of HBV virus replication in the supernatant was 2.37 卤0.22,3.50 卤0.27,6.02 卤0.56, 17.74 卤0.96, respectively, and there was significant difference among the three groups (F = 20.726, P < 0.001). Similarly, Hep G2-HBV1.1 cells were treated with 0, 0.1, 0.2 and 0.5 渭 mol / L Epiromycin, respectively, and the amount of HBV virus replication in the supernatant was 3.75 卤0.35,6.05 卤0.48,10.28 卤1.01,20.86 卤1.81, respectively, and that in the supernatant was 3.75 卤0.35,6.05 卤0.48,10.28 卤1.01,20.86 卤1.81, respectively. The relative amount of HBV DNA replication intermediates in the supernatant of Hep G2.2.15 cells was 1.08 卤0.02 and 2.69 卤0.05 in the control group and the drug treatment group, respectively, and the relative amount of HBV DNA replication intermediates in the supernatant of Hep G2.2.15 cells was 1.08 卤0.02 and 2.69 卤0.05 in the control group and the drug treatment group, respectively. There was a significant difference between the control group and the drug treatment group (t = 58.33, P < 0.000). Similarly, the relative amount of HBV DNA replication intermediates in Hep G2-HBV1.1 cells was 1.03 卤0.01 in the control group and 3.52 卤0.05 in the drug treatment group, and there was a significant difference between the control group and the drug treatment group (t = 85.801, P < 0.001). These results suggest that Epiromycin promotes the secretion or release of more HBV into the supernatant of cells. Conclusion: Epiromycin can directly promote the replication of HBV, which may be a new mechanism of HBV reactivation after chemotherapy.
【作者单位】：重庆医科大学附属第一医院检验科;重庆医科大学病原生物学教研室;重庆医科大学感染性疾病分子生物学教育部重点实验室;
【基金】：国家临床重点专科建设经费资助项目【编号:(财社2010)305号】
【分类号】：R96

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