反义寡核苷酸抗乙型肝炎病毒表达诱导凋亡的体外研究

In vitro study of antisense oligodeoxynucleotides to inhibit HBV expression and induce apoptosis

目的 研究反义脱氧寡核苷酸 (ASON)抗乙型肝炎病毒 (HBV)复制表达的作用。方法 设计合成针对HBVPreS2 的ASON并设非互补序列作对照 ,以人肝癌细胞系 2 .2 .15细胞为细胞模型 ,应用ELISA观察了ASON对HBV基因表达的抑制作用 ,流式细胞仪观察了ASON对宿主细胞凋亡和增殖的影响。应用RIA法和MTT法观察ASON对细胞代谢的影响。结果 ASON可有效地抑制HBV基因的表达 ,对HBsAg和HBeAg的抑制率分别为 6 6 %和 91% ,而非互补序列对照无抑制作用 ;ASON可诱导宿主细胞凋亡 ,用药后第 3天和第 6天凋亡率分别为 6 .10 %和 6 .43% ,增殖指数分别为 37%和 36 % ,RIA和MTT法表明ASON对宿主细胞无毒性作用。结论 ASON不仅以序列特异性方式发挥抗病毒作用 。

Objective To study the effect and mechanism of antisense oligodeoxynucleotides (ASONs) inhibiting hepatitis B virus (HBV) expression. Methods We designed and synthesized antisense oligodeoxynucleotides directly against HBV PreS 2 gene and noncomplementary sequence control. 2.2.15 cells were chosen as cell model. Inhibitory effect of ASONs on HBV gene expression were assayed by ELISA. Cell apoptosis and proliferation were detected by Fascan Flow Cell Cytometer. Effect of ASONs on cell metabolism was detected by radioimmunoassay (RIA) and MTT assay. Results ASONs were able to effectively inhibit HBV expression. Their inhibitory rates of HBsAg and HBeAg were 66% and 91%, respectively. Noncomplementary sequence control group (both inhibitory rates were 11%) was not able to inhibit HBV expression. ASON might induce host cell apoptosis. Cell apoptosis rates on 3rd and 6th day were 6.10% and 6.43%, respectively. Proliferation index were 37% and 36%, respectively. Results of RIA and MTT showed that ASON had not cytoxicity on host cell. Conclusions Not only are ASON able to inhibit HBV gene expression with sequence specific but also clear HBV in the way of apoptosis.