反义核酸对癌基因C-myc和N-ras蛋白表达的影响

EFFECT ON C-myc AND N-ras EXPRESSION OF ANTISENCE OLIGODEOXYNUCLEOTIDES

目的 :为了研制新型的抗肝癌药物 ,观察了反义寡核苷酸对整合乙型肝炎病毒 (HBV)的肝癌细胞基因治疗的作用。方法 :设计合成针对 HBV Pre S2基因翻译起始位点的反义寡核苷酸 (ASON) ,并设非互补序列作对照 ,选择 2 .2 .15细胞为细胞模型 ,ASON以 10 μmol/ L 浓度用药 ,用 EL ISA法观察了细胞培养上清液中 HBs Ag和 HBe Ag的变化 ,MTT法检测 ASON对细胞的毒性作用 ,放射免疫方法检测细胞培养上清液中血清铁蛋白浓度。免疫细胞化学染色检测 ASON对癌基因 C- m yc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 HBs Ag和 HBe Ag的抑制率分别为 6 6 %和 91%。而 ASON以 2 0 μm ol/ L 或 40 μmol/ L 浓度用药 4天对宿主细胞无毒性作用 ,ASON以 10 μmol/ L 浓度用药后第二天用药组和对照组血清铁蛋白浓度分别为 (2 8.2 6± 0 .0 2 ) μg/ L 和 (2 8.14± 0 .0 2 ) μg/ L,两者相比差异无显著性 (P>0 .0 5 )。用药后第 3天 C- myc和 N- ras蛋白表达降低 ,和对照组相比差异有显著性 (P<0 .0 5 )。结论 :该段反义寡核苷酸不仅抑制 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 make new drugs for hepatocellular carcinoma,the effect of antisence oligodeoxynucleotides on hepatocellular carcinoma cell line was studied.Methods:We designed and synthesized antisense oligodeoxynucleotides directed against HBV PreS2 gene and noncomplementary sequence control.2.2.15 cells were chosen as cell model.Inhibitory effect of ASON inhibiting HBV gene expression were assayed by ELISA.The cytotoxicity experiment of ASON were detected by MTT assay.Effect of ASON on serum ferritin expression of host cell were detected by radioimmunoassay(RIA).The effect of ASON on C myc and N ras protein expression of 2.2.15 were measured with ABC.Rusults:ASON were able to inhibit HBV gene expression.The inhibitory rates of HBsAg and HBeAg were 66 % and 91 %,respectively.ASONs at 20 μmol/L or 40 μmol/L concentration had not any cytoxicity on cells .Serum ferritin concentration were (28.26±0.02)mg/L in ASON group and (28.14±0.02)mg/L in control group.There was not a significant difference between ASON group and control group( P >0.05).The C myc and N ras protein expression of 2.2.15 were reduced after ASONs were used 3 days.There was a significant difference between ASON group and control group ( P <0.05).Conclusion:ASON can not only inhibit HBV gene expression,but also inhibit some carcinoma phenotype.We think ASON directed against HBV PreS2 has a vast protential for treatment of hepatocellular carcinoma .