反义寡核苷酸体外抗乙型肝炎病毒研究

Study on anti-HBV effects by antisense oligodeoxynucleotides in vitro

目的 探讨反义寡核苷酸的体外抗病毒作用 ,为基因水平上防治乙型肝炎病毒 (HBV)感染及HBV相关肝细胞癌的基因治疗提供实验依据。方法 用PCR方法分别合成了互补于HBVX基因的翻译起始区、DR2区、ENⅡ区的反义寡核苷酸 (AsON)及无关序列 ,其中针对ENⅡ设计的AsON ,目前尚未见互补于该区段反义寡核苷酸的报道。以HBVDNA转染的HepG2 2 15细胞为靶细胞 ,ELISA法检测AsON作用前后细胞上清中的HBsAg和HBeAg分泌情况。结果 当AsON的最佳作用浓度为 10 μmol L时 ,3种反义寡核苷酸对HBsAg和HBeAg抑制率分别为 :5 7%、6 0 %、5 2 %和 5 6 %、45 %、5 6 % ,AsON对HBV抗原的抑制作用具双峰现象。无关序列无明显抑制作用 (<12 % )。利用锥虫蓝染色、四甲基偶氮唑盐比色试验观察到当AsON作用浓度为 40 μmol L时 ,对细胞代谢无明显毒副作用。结论 反义寡核苷酸封闭HBVX基因区的关键区段 ,可有效抑制HBV抗原的分泌。

Objective To study the inhibition effect of anti-HBV oligodeoxy-nucleotides on HBV X gene.Methods Three antisense phosphorothioate oligodeoxy-nucleotides(AsON) complementary to the initiator of X 、DR2、ENⅡrespectively, were synthesized and tested for their anti-HBV activity on HepG2.2.15 cells with ELISA methods. Results When the best effect concentration of AsON was 10 μmol/L, the inhibition rates on HBsAg of three AsON were 57%, 60% and 52% respectively, and on HBeAg were 56%, 45% and 56% respectively. There were two inhibition peaks at different times. There was no inhibition effect treated by random control (<12%). Using the 3-(4,5-dimethythiazol-zyl)-2,5-diphnyl tetrazolium bromide method, there was no cytotoxicity at 40 μmol/L of AsON. Conclusion AsONs on three key region of X gene were effective drugs to inhibit the expression of HBV.