反义寡核苷酸体外抗乙型肝炎病毒研究

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HBV X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HBV DNA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10μmol/L时,3种反义寡核苷酸对HBsAg和HBeAg抑制率分别为;57％、60％、52％和56％、45％、56％,AsON对HBV抗原的凶制作用具双峰现象。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40μmol/L时,对细胞代谢无毒副作用。结论反义寡核苷酸封闭HBV X基因区的关键区段,可有效抑制HBV抗原的分泌。

Study on anti-HBV effects by antisense oligodeoxynucleotides in vitro

OBJECTIVE: To study the inhibition effect of anti-HIV oligodeoxy-nucleotides on HBV X gene. METHODS: Three antisense phosphorothioate oligodeoxy-nucleotides(AsON) complementary to the initiator of X, DR2, EN II respectively, 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 mumol/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-xyl)-2,5-diphnyl tetrazolium bromide method, there was no cytotoxicity at 40 mumol/L of AsON. CONCLUSION: AsONs on three key region of X gene were effective drugs to inhibit the expression of HBV.