preS2反义寡核苷酸降低肝癌细胞HepG2.2.15的致瘤性

Suppression of tumorigenicity of HepG2.2.15 hepatocellular carcinoma cell line by HBpreS2 antisense therapy

目的 :观察反义寡核苷酸 (as preS2 )对HepG2 .2 .15细胞致瘤的抑制作用。方法 :合成针对preS2翻译起始区反义寡核苷酸 ,以 10 μmol L的终浓度作用于HepG2 .2 .15细胞 ,用FACSCAN流式细胞仪检测细胞凋亡率 ,TRAP银染测定细胞端粒酶活性 ,用ABC免疫化学染色P2 1ras、P6 2 c myc蛋白 ;同时收集上清以RIA法检测血清铁蛋白浓度 ,ELISA法测定HBsAg、HBeAg浓度。体内抑瘤实验以HepG2 .2 .15细胞荷瘤裸鼠进行。结果 :as preS2可显著抑制HBsAg、HBeAg表达 ,抑制率分别为 6 6 %和 91%。as preS2作用 3d后抑制P2 1ras、P6 2 c myc蛋白表达 ,并降低肝癌细胞端粒酶活性 ,同时明显诱导细胞凋亡 (6 .13%vs 1.16 % ,P <0 .0 5 )。as preS2不影响宿主细胞自身血清铁蛋白的分泌。体外在荷HepG2 .2 .15细胞裸鼠中注射as preS2可抑制成瘤率。结论 :as preS2在体内外均能有效抑制HepG2 .2 .15细胞增殖 ,as preS2不仅抑制HBV抗原表达 ,而且明显降低肝癌细胞端粒酶活性 ,诱导肝癌细胞凋亡。

Objective:To observe the effect of antisense oligodeoxynucleotide targeting to HBpreS2 (as preS2) on tumorigenicity of hepatocellular carcinoma cell line HepG2.2.15 both in vitro and in vivo.Methods:HepG2.2.15 cells were incubated in the presence of as preS2(10?μmol/L) and were collected and assayed for apoptosis,telomeras activity and expression of oncogenes(P21 ras and P62 c myc )by flow cytometry ,TRAP method and immunochemical staining respectively.The supernatant was collected to detect the expression of HBsAg ,HBeAg and the concentration of serum ferritin by ELISA and RIA.Inhibitory effect of as preS2 in vivo was tested in nude mice bearing HepG2.2.15 cells.Results:As preS2 had a distinctive inhibitory effect on HBV gene expression of HBsAg and HBeAg (66% and 91% respectively). As preS2 decreased the expression of P21 ras and P62 c myc ,as well as the telomerase activity in HepG2.2.15 cells. At the same time,6.13% of cells treated with as preS2 underwent apoptosis,while 1.16% of control cells underwent programmed cell death. However, serum ferritin concentration in supernatant of HepG2.2.15 had no significant difference in as preS2 treated group and the control group (P>0.05).When HepG2.2.15 cells were implanted into nude mice,injection of as preS2 could significantly inhibited the tumorigenicity of HepG2.2.15 cells.Conclusion:As preS2 decreased the tumorigenicity of HepG2.2.15 cells both in vivo and in vitro.It could not only inhibit the HBV gene expression, but also decrease the telomerase activity and induce the apoptosis of HepG2.2.15.