摘要
目的:研制新型的抗肝癌药物,观察反义脱氧寡核苷酸(asON)对整合乙型肝炎病毒(HBV)的HepG2.2.15细胞系端粒酶活性及癌基因C-myc和N-ras蛋白表达的影响。方法:设计合成针对HBVPreS2基因翻译起始区的asON,并设非互补序列作对照,以肝癌细胞系HepG2.2.15为细胞模型,asON以10μmol/L浓度用药,用ELISA法观察了细胞培养上清液中HBsAg和HBeAg的变化;MTT法检测asON对细胞毒性作用;放射免疫方法检测细胞培养上清液中血清铁蛋白浓度;用银染—Trap法测定asON对HepG2.2.15细胞端粒酶活性的影响;免疫细胞化学染色检测asON对癌基因C-myc和N-ras蛋白表达的影响。结果:asON能有效地抑制HBV抗原表达,对HBsAg和HBeAg的抑制率分别为66%和91%。而asON以20或40μmol/L浓度用药4d对宿主细胞无毒性作用,asON以10μmol/L浓度用药后第2天用药组和对照组血清铁蛋白浓度分别为(28.26±0.02)ng/ml和(28.14±0.02)ng/ml,两者相比无统计学意义(P>0.05)。用药后第3天asON组比对照组端粒酶活性降低;用药后第3天C-myc和N-ras蛋白表达降低,和对照组相比有统计学意义(P<0.05)。结论:该段asON不仅抑制HBV抗原表达,而且抑制肝癌细胞的恶性表型而对宿主细胞无毒性,有可能成为有前途的抗肝细胞肝癌新药。
Objective:In order to prepare new drugs of therapy for hepatocellular carcinoma,we in vestigated the effect of antisense oligodeoxynu cleotides(ASON)directed against HBV PreS 2 ini tiator on telom erase activity,C-myc and N-ras protein expression of hepatocellular carcinoma cell line HepG2.2.15.Methods:We designed and synthesized ASON directed against HBV PreS 2 gene initiator and non-comple men tary se quence control.HepG2.2.15cells were chosen as cell model.In hibitory effect of ASON in hibiting HBV gene expression were assayed by ELISA.The cytotoxicity experiment of ASON had been detected by MTT assay.Effect on serum ferritin expression of host cell in the presence of ASON were detected by ra dioim munoassay(RIA).The effect on the telom erase activity of HepG2.2.15in the presence of ASON were assayed by trap methods.The effect of ASON on the C-myc and N-ras protein expression of HepG2.2.15were measured by ABC.Rusults:ASON at10μmol/L concentration were able to effectively inhibit HBV gene expression HBsAg and HBeAg.The inhibitory rates of HBsAg and HBeAg were66%and91%,re spectively.ASON at20μmol/L or40μmol/L concentration hadn't any cytotox icity on host cells.Serum ferritin con centra tion were(28.26±0.02)ng/ml in ASON group and(28.14±0.02)ng/ml in con trol group,re spectively.There was not a sig-nificant difference between ASON group and control group(P>0.05).ASON re duced the telomerase ac tivity of HepG2.2.15cells.The C-myc and N-ras protein expression of HepG2.2.15were re duced after ASON were used3days.There was a significant difference between ASON group and control group(P<0.05).Conclusion:Not only were ASON able to specific in hibit HBV gene ex pression but also inhibit some carcinoma phenotype.We think A-SON directed against HBV PreS 2 transla tion initiator have a vast prospect for treatment of hepatocellular carcinoma patients chronically infected with HBV.
出处
《山东大学学报（医学版）》
CAS
2003年第1期52-55,共4页
Journal of Shandong University:Health Sciences
基金
山东省医药卫生"九五"攻关课题(9734)
国家自然基金资助项目(39970333)
关键词
反义寡核苷酸类
乙型肝炎病毒
肝细胞癌
基因治疗
端粒酶
Oligodeoxynu cleotides,antisense
Hepatitis B virus
Carcinoma,hepatocellu lar
Gene therapy
Telom erase
