10-23脱氧核酶靶向抑制喉癌eIF4E基因表达的实验研究

Research of 10-23 DNAZyme inhibit the expression of eIF4E genes

目的:在细胞水平上探讨10-23脱氧核酶(DNAzyme)成为新型喉癌基因治疗药物的可能性。方法:设计合成针对原癌基因eIF4E mRNA编码区的1059位点的10-23DNAzyme,并对其进行硫代磷酸化修饰,经脂质体转染Hep-2细胞,倒置相差荧光显微镜观察Hep-2细胞对5-FAM荧光素标记10-23DNAzyme的摄取,流式细胞仪计算转染效率,半定量RT-PCR、western blot观察其对喉癌eIF4E基因表达的抑制效应。结果:倒置相差荧光显微镜观察在转染12、24、36、48h后,阳性转染的细胞均可发出绿色荧光,流式细胞仪检测转染效率分别为60%、85%、90%、89%;修饰的10-23DNAzyme作用于Hep-2细胞后可显著抑制eIF4E基因的表达,明显低于作为对照的转染空脂质体组和Hep-2细胞组(均P<0.05)。结论:经过硫代化修饰的10-23DNAzyme在细胞水平上能高效阻断eIF4E基因的表达,是一种高度特异性的、高效的基因治疗剂。

Objective：To explore the possibility of 10-23DNAzyme becoming a new gene therapy for laryngeal carcinoma treatment at the cell level. Method： Thosthorothioate 10-23DNAzym especific to eIF4E gene mRNA 1059 was designed and synthesized, and its inhibition effects on the expression of eIF4E gene in Hep-2 cells were observed. Result： The expression of eIF4E gene was remarkable depressed after Hep-2 cells was transfected by DNAzyme. The level of inhibiting eIF4E in hep-2 cells transfected by DNAzyme was lower than that by only lipofectimine2000 transfected and Hep-2. Conclusion： The expression of eIF4E gene in Hep-2 cells 10-23DNAzyme can be highly blocked. It is a specific and effective gene therapeutic means.