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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 10-23 deoxyribonuclease (DNAzyme) becoming a novel gene therapy for laryngeal cancer at the cellular level. METHODS: The 10-23 DNAzymes targeting the 1059 site of eIF4E mRNA coding region were designed and synthesized. The thiophosphorylation was performed. Hep-2 cells were transfected by liposome and Hep-2 was observed by inverted phase contrast fluorescence microscope. The uptake of 5-FAM fluorescein-labeled 10-23 DNAzyme into cells was measured by flow cytometry. Semi-quantitative RT-PCR and western blot were used to observe the inhibitory effect of eIF4E on laryngeal cancer eIF4E expression. RESULTS: Inverted phase contrast fluorescence microscopy showed that positive transfected cells could emit green fluorescence after 12, 24, 36, and 48 hours of transfection, and the transfection efficiency was 60%, 85%, 90%, and 89 respectively by flow cytometry. %; The modified 10-23 DNAzyme significantly inhibited the expression of eIF4E gene in Hep-2 cells, which was significantly lower than that of the control transfected liposome group and Hep-2 cell group (all P<0.05). Conclusion: The thiolated 10-23 DNAzyme can effectively block the expression of eIF4E gene at the cellular level. It is a highly specific and efficient gene therapy agent.