α-鹅膏毒肽对乙型肝炎病毒复制的体外影响
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摘要
目的：基于α-鹅膏毒肽(α-amanitin)的作用机制和乙肝病毒复制转录特点，初步探讨α-鹅膏毒肽对HepG2.2.15细胞的毒性及其对乙型肝炎病毒(HBV)复制的影响。
方法：采用MTT法检测不同浓度α-鹅膏毒肽对体外培养的HepG2 2.2.15细胞株的毒性，然后在无毒浓度下用不同浓度α-鹅膏毒肽或拉米夫定与HepG2 2.2.15细胞共培养6天，分别在第3天和第6天收集细胞培养上清液，采用时间分辨免疫荧光法(TRFIA)测定上清HBsAg、HBeAg浓度；收获细胞提取总RNA，行逆转录聚合酶链反应(RT-PCR)检测细胞内HBsAg和HBcAg mRNA的表达。
结果：①在0.0064μg／ml～0.8μg／mlα-鹅膏毒肽时，对HepG22.2.15细胞无明显毒性作用，当浓度达到4μg／ml～20μg／ml时毒性较明显，第3天TC_(50)为10.190μg／ml，第6天TC_(50)为8.471μg／ml；
②对上清夜HBsAg的影响：第3天，3TC组、0.8μg／ml组和0.16μg／ml组对HBsAg抑制率分别为46.071％、19.881％和15.952％，其余各组未见抑制作用；第6天前述三组的抑制率分别为55.709％、47.405％和29.296％，均P＜0.01，其余各组抑制作用不明显；
③对上清夜HBeAg的影响：第3天3TC组和0.8μg／ml组对HBsAg抑制率分别为31.858％和14.159％，其余各组未见抑制作用；第6天3TC组、0.8μg／ml组、0.16μg／ml组和0.032μg／ml组α-amanitin浓度组对HBeAg抑制率分别59.161％、51.524％、45.845％和29.086％。0.0064μg／mlα-amanitin组抑制作用不明显；
④对胞内HBsAg mRNA表达水平的影响：3TC组、0.8μg／ml组、0.16μg／ml组和0.032μg／ml组3个α-amanitin浓度组随浓度的增加，表达逐渐下调，第3天，3TC组、0.8μg／ml组与其他各组比较差异有显著性，第6天，以上四组与对照组相比差异有显著性，其余各组未见明显差异；
⑤对胞内HBcAg mRNA表达水平的影响：第3天，3TC组、0.8μg／ml组HBcAg mRNA表达下调，与对照组比较差异有显著性，其余各组未见明显差异；第6天，3TC组、0.8μg／ml组、0.16μg／ml组与对照组相比差异均有显著性。
⑥第6天与第3天相比，3TC组、0.8μg／ml组和0.16μg／ml组细胞内HBsAg、HBcAg mRNA表达下调，上清液中HBsAg、HBeAg浓度也随之下降；其中3TC组抑制作用最明显，实验组以0.8μg／ml组最明显。
结论：在本实验条件下α-鹅膏毒肽在0.0064μg／ml～0.8μg／ml浓度时对HepG2 2.2.15细胞的毒性较小，低毒浓度下α-鹅膏毒肽可抑制HepG2 2.2.15细胞HBeAg、HBsAg的分泌及HBsAg和HBcAg mRNA的表达。随浓度的增加，抑制作用越明显，说明α-鹅膏毒肽对乙型肝炎病毒复制有一定程度的抑制作用。
Objective To explore the roles ofα-amanitin in the replication ofhepatitis B virus in HepG2 2.2.15 cell line
Methods MTT assay were used to observe the cytotoxicity ofα-amanitin to the HepG 2 2.2.15 cell line. And then HepG2.2.15 cellswere cultured with different non-cellulotoxic concentration ofα-amanitin or 3TC for six days. TRFIA was adopted to determine thelevels of HBsAg and HBeAg,after the cell culture medium was collectedin the third day and the sixth day,respectively. Cells were harverested fordetermining the expression of HBsAg mRNA and HBcAg mRNA byreverse transcription PCR (RT-PCR)
Results①α-amanitin has no significant cytotoxicity to HepG22.2.15 cell line in the concentration ranging from 0.0064μg/ml to0.8μg/ml,while significant cytotoxicity was found in the concentrationranging from 4μg/ml to 20μg/ml. In the third day, TC_(50) was 10.190μg/ml,in the sixth day, TC_(50) was 8.471μg/ml.
②In the third day, HBsAg suppression rate of 3TC, 0.8μg/ml and0.016μg/ml group was 46.071%、19.881% and 15.952%,respectively.No suppression was found in other groups.In the sixth day,HBsAg suppression rate of 3TC, 0.8μg/ml and 0.016μg/ml group was55.709%、47.405% and 29.296%, respectively.No suppression was foundin other groups.
③In the third day, HBeAg suppression rate of 3TC and 0.8μg/mlgroup was 31.854% and 14.159%, respectively.No suppression wasfound in other groups.In the sixth day, HBsAg suppression rate of 3TC,0.8μg/ml、0.16μg/ml and 0.032μg/ml group was 59.161%、51.524%、 45.845% and 29.086%, respectively.No suppression was found in0.0064μg/ml group.
④In the third day, down regulation of HBsAg mRNA expression in3TC, 0.8μg/ml and 0.16μg/ml groups was found statistical significance,comparing with the control.In the sixth day, down regulation of HBsAgmRNA expression in 3TC, 0.8μg/ml and 0.16μg/ml groups was foundstatistical significance, comparing with the control group.
⑤In the third day, down regulation of HBcAg mRNA expression in3TC and 0.8μg/ml groups was found statistical significance, comparingwith the control group.In the sixth day, down regulation of HBsAgmRNA expression in 3TC, 0.8μ/ml and 0.16μg/ml groups was foundstatistical significance, comparing with the control group.
Conclusionsα-amanitin has no significant cytotoxicity to HepG22.2.15 cell line in the concentration ranging from 0.0064μg/ml to0.8μg/ml,while significant cytotoxicity was found in the concentrationranging from 4μg/ml to 20μg/ml. In the third day, TC_(50) was 10.190μg/ml,in the sixth day, TC_(50) was 8.47μg/ml. Both the expression ofHBsAg and HBcAg mRNA and the secretion of HBsAg and HBeAg weresuppressed byα-amanitin at little cytotoxicity concentrtion in HepG22.2.15 cell line.Its suggested thatα-amanitin might inhibit theduplication of HBV-DNA,being provided therical and experimentalevidence for the further research of the pharmacological mechanism.
方法：采用MTT法检测不同浓度α-鹅膏毒肽对体外培养的HepG2 2.2.15细胞株的毒性，然后在无毒浓度下用不同浓度α-鹅膏毒肽或拉米夫定与HepG2 2.2.15细胞共培养6天，分别在第3天和第6天收集细胞培养上清液，采用时间分辨免疫荧光法(TRFIA)测定上清HBsAg、HBeAg浓度；收获细胞提取总RNA，行逆转录聚合酶链反应(RT-PCR)检测细胞内HBsAg和HBcAg mRNA的表达。
结果：①在0.0064μg／ml～0.8μg／mlα-鹅膏毒肽时，对HepG22.2.15细胞无明显毒性作用，当浓度达到4μg／ml～20μg／ml时毒性较明显，第3天TC_(50)为10.190μg／ml，第6天TC_(50)为8.471μg／ml；
②对上清夜HBsAg的影响：第3天，3TC组、0.8μg／ml组和0.16μg／ml组对HBsAg抑制率分别为46.071％、19.881％和15.952％，其余各组未见抑制作用；第6天前述三组的抑制率分别为55.709％、47.405％和29.296％，均P＜0.01，其余各组抑制作用不明显；
③对上清夜HBeAg的影响：第3天3TC组和0.8μg／ml组对HBsAg抑制率分别为31.858％和14.159％，其余各组未见抑制作用；第6天3TC组、0.8μg／ml组、0.16μg／ml组和0.032μg／ml组α-amanitin浓度组对HBeAg抑制率分别59.161％、51.524％、45.845％和29.086％。0.0064μg／mlα-amanitin组抑制作用不明显；
④对胞内HBsAg mRNA表达水平的影响：3TC组、0.8μg／ml组、0.16μg／ml组和0.032μg／ml组3个α-amanitin浓度组随浓度的增加，表达逐渐下调，第3天，3TC组、0.8μg／ml组与其他各组比较差异有显著性，第6天，以上四组与对照组相比差异有显著性，其余各组未见明显差异；
⑤对胞内HBcAg mRNA表达水平的影响：第3天，3TC组、0.8μg／ml组HBcAg mRNA表达下调，与对照组比较差异有显著性，其余各组未见明显差异；第6天，3TC组、0.8μg／ml组、0.16μg／ml组与对照组相比差异均有显著性。
⑥第6天与第3天相比，3TC组、0.8μg／ml组和0.16μg／ml组细胞内HBsAg、HBcAg mRNA表达下调，上清液中HBsAg、HBeAg浓度也随之下降；其中3TC组抑制作用最明显，实验组以0.8μg／ml组最明显。
结论：在本实验条件下α-鹅膏毒肽在0.0064μg／ml～0.8μg／ml浓度时对HepG2 2.2.15细胞的毒性较小，低毒浓度下α-鹅膏毒肽可抑制HepG2 2.2.15细胞HBeAg、HBsAg的分泌及HBsAg和HBcAg mRNA的表达。随浓度的增加，抑制作用越明显，说明α-鹅膏毒肽对乙型肝炎病毒复制有一定程度的抑制作用。
Objective To explore the roles ofα-amanitin in the replication ofhepatitis B virus in HepG2 2.2.15 cell line
Methods MTT assay were used to observe the cytotoxicity ofα-amanitin to the HepG 2 2.2.15 cell line. And then HepG2.2.15 cellswere cultured with different non-cellulotoxic concentration ofα-amanitin or 3TC for six days. TRFIA was adopted to determine thelevels of HBsAg and HBeAg,after the cell culture medium was collectedin the third day and the sixth day,respectively. Cells were harverested fordetermining the expression of HBsAg mRNA and HBcAg mRNA byreverse transcription PCR (RT-PCR)
Results①α-amanitin has no significant cytotoxicity to HepG22.2.15 cell line in the concentration ranging from 0.0064μg/ml to0.8μg/ml,while significant cytotoxicity was found in the concentrationranging from 4μg/ml to 20μg/ml. In the third day, TC_(50) was 10.190μg/ml,in the sixth day, TC_(50) was 8.471μg/ml.
②In the third day, HBsAg suppression rate of 3TC, 0.8μg/ml and0.016μg/ml group was 46.071%、19.881% and 15.952%,respectively.No suppression was found in other groups.In the sixth day,HBsAg suppression rate of 3TC, 0.8μg/ml and 0.016μg/ml group was55.709%、47.405% and 29.296%, respectively.No suppression was foundin other groups.
③In the third day, HBeAg suppression rate of 3TC and 0.8μg/mlgroup was 31.854% and 14.159%, respectively.No suppression wasfound in other groups.In the sixth day, HBsAg suppression rate of 3TC,0.8μg/ml、0.16μg/ml and 0.032μg/ml group was 59.161%、51.524%、 45.845% and 29.086%, respectively.No suppression was found in0.0064μg/ml group.
④In the third day, down regulation of HBsAg mRNA expression in3TC, 0.8μg/ml and 0.16μg/ml groups was found statistical significance,comparing with the control.In the sixth day, down regulation of HBsAgmRNA expression in 3TC, 0.8μg/ml and 0.16μg/ml groups was foundstatistical significance, comparing with the control group.
⑤In the third day, down regulation of HBcAg mRNA expression in3TC and 0.8μg/ml groups was found statistical significance, comparingwith the control group.In the sixth day, down regulation of HBsAgmRNA expression in 3TC, 0.8μ/ml and 0.16μg/ml groups was foundstatistical significance, comparing with the control group.
Conclusionsα-amanitin has no significant cytotoxicity to HepG22.2.15 cell line in the concentration ranging from 0.0064μg/ml to0.8μg/ml,while significant cytotoxicity was found in the concentrationranging from 4μg/ml to 20μg/ml. In the third day, TC_(50) was 10.190μg/ml,in the sixth day, TC_(50) was 8.47μg/ml. Both the expression ofHBsAg and HBcAg mRNA and the secretion of HBsAg and HBeAg weresuppressed byα-amanitin at little cytotoxicity concentrtion in HepG22.2.15 cell line.Its suggested thatα-amanitin might inhibit theduplication of HBV-DNA,being provided therical and experimentalevidence for the further research of the pharmacological mechanism.
引文
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[2] Liaw YF. Therapy of chronic hepatitis B: current challenges and opportunities. J Viral Hepat, 2002,9:393-399.
[3] Morgan M, Park W, Keeffe EB. Diagnosis and treatment of chronic hepatitis B: an update. Minerva Gastroenterol Dietol, 2007, 53(1):25～41.
[4] Andres Ruiz-Sancho, Julie Sheldon, Vincent Soriano. Telbivudine: a new option for the treatment of chronic hepatitis B. Expert Opin Biol Ther, 2007, 7(5): 751～761
[5] Rivkin A. Entecavir: A new nucleoside analogue for the treatment of chronic hepatitis B. Drugs Today (Barc), 2007, Apr, 43(4):201～220.
[6] Perrillo RP, Schiff ER. Davis GL, et al. A randomized, controlled trial of interferon alpha-2b alone and after prednisone withdrawal for the treatment of chronic hepatitis B. N Engl J Med, 1990, Aug 2, 323(5):295～301
[7] Schalm SW, Van Rossum JGJ. Goals of antiviral therapy:Viral clearance or ALT normalization. Hepatol clin, 1998, 6(suppl1):85～91
[8] Hosaka T, Suzuki F, Suzuki Y, et al. Factors associated with the virological response of lamivudine-resistant hepatitis B virus during combination therapy with adefovir dipivoxil plus lamivudine. J Gastroenterology, 2007, 42(5): 368～374
[9] Trojan J, Stuermer M, Teuber G, et al. Treatment of patients with lamivudine-resistant and adefovir dipivoxil-resistant chronic hepatitis B virus infection: is tenofovir the answer?. Gut, 2007, 56(3):436-437
[10] 成军，杨守纯。现代肝炎病毒分子生物学．北京：人民军医出版社，1997
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