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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
Objective To study the two 19-base ASS ODN (AS1 and AS2) pairs complementary to the start codon site of the primary immediate early phase (MIE) mRNA of CMV and the intron 1 / exon 2 splice site of the MIE mRNA precursor Inhibition of CMV replication. Methods The amount of CMV antigen on 2BS cells was measured by in situ ELISA. Results Both S ODNs had anti-CMV effect. The median effective concentrations (EC50) were 453 and 102 μmol·L-1, respectively. AS1 at 80 μmol·L-1 delayed CMV antigen secretion by 3-4 days. The effect of dosing immediately after infection was the best, and the dosing effect dropped significantly after 12h (P <0.05). The same dosage was added 36h after infection and the combination with DHPG was significantly enhanced (P <0.05). The two kinds of ASS ODN 6 4 0 μmol·L-1 had only slight cytotoxicity, and the concentrations below 160 μmol·L-1 had no obvious side effects. The liposomes with 5 mg · L -1 reduced the EC50 of the two S ODNs by 111 0 and 151.7 times, respectively. The two justice sequence controls also showed inhibitory effects with EC50 of 2 6 2 and 30 1 μmol·L -1, respectively. NorthernBlot analysis suggests that activation of RNase H degrades mRNA molecules in hybrid duplexes as an important specific mechanism of action, which interferes with the nonspecific mechanism of CMV adsorption and invasion into cells. Conclusions ASS ODN acting on MIE gene can effectively inhibit the replication of CMV and is worth further study for the treatment of CMV infection