反义寡聚硫代磷酸脱氧核糖核苷酸的抗巨细胞病毒作用

目的　研究互补于CMV主要即刻早期 (MIE)mRNA的起始密码子位点和MIEmRNA前体内含子 1/外显子 2拼接位点的 2个 19个碱基的ASS ODN (AS1和AS2 )对CMV复制的抑制作用。方法　采用原位ELISA法测定 2BS细胞上CMV抗原量。结果　 2个S ODN均有抗CMV作用 ,半数有效浓度 (EC50 )分别为 4 5 3和 10 2 μmol·L-1。 8 0 μmol·L-1的AS1使CMV抗原分泌推迟 3～ 4d。感染后立即加药效果最好 ,12h后加药效果大大下降 (P <0 0 5 ) ,感染后 36h再添加同一剂量及与DHPG联合使用效果明显增强 (P <0 0 5 )。 2种ASS ODN 6 4 0 μmol·L-1时仅有轻微的细胞毒性 ,16 0 μmol·L-1以下浓度无明显毒副作用。采用 5mg·L-1的脂质体使 2种S ODN的EC50 分别降低了 111 0和 15 1 7倍。 2种正义序列对照也显示出抑制效应 ,EC50 分别为 2 6 2和 30 1μmol·L-1。NorthernBlot分析提示激活RNA酶H降解杂交双链中的mRNA分子为重要的特异性作用机制 ,而干扰CMV对细胞的吸附与侵入为重要的非特异性作用机制。结论　作用于MIE基因的ASS ODN可有效地抑制CMV复制 ,值得进一步研究以应用于治疗CMV感染

Anti-cytomegalovirus (CMV) effect of antisense oligodeoxynucleotidesphosphorothioates (AS S-OND)

AIM To study anti CMV effect of S ODNs complementary to the initial code region of major immediate early (MIE) mRNA, and to the intron1/exon2 boundary of MIE mRNA precursor. METHODS To evaluate the anti CMV effect by determining viral antigen on infected 2BS cells by in situ ELISA. RESULTS Both of 2 AS S ODNs and their sense sequence control showed anti CMV effect. The medium effective concentration (EC 50 ) were 4 53, 10 2, 26 2 and 30 1 μmol·L -1 . The secretion of CMV antigens were delayed by 3 d～4 d by 8 0 μmol·L -1 AS 1. It showed increased effect by administrating earlier postinfection, by supplementing the S ODN at intervals, by conbinating with ganciclovir, and by packaging with liposome. A slight cytotoxicity was observed at the concentration of 64 0 μmol·L -1 . Northern blot analysis indicated that the MIE mRNA decreased after the treatment of AS 1. It suggests that disintegration of the mRNA in the hybridized duplex by activated RNase H played an important role as the mechanism of specific action, and "time and effect" analysis suggested that interference of viral adsorbtion and penetration may be the important mechanism of nonspecificity. CONCLUSION AS S ODNs targeted to MIE gene are effective anti CMV agents which can be developed as a new type of chemotherapy drugs against CMV infection.