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目的 探讨反义肽核酸(PNA)的细胞转染方法及对体外培养的神经母细胞瘤多药耐药性(MDR)的逆转作用。方法 以人MDR 1基因mRNA为靶点设计合成两反义PNA序列,利用PNA DNA杂交,阳离子脂质体介导转染神经母细胞瘤耐药细胞株SK N SH。应用流式细胞术、RT-PCR、MTT等方法分别检测反义PNA的转染效率、转染前后细胞P 糖蛋白(P gp)、MDR 1基因mRNA的表达以及对化疗药物的敏感性。结果 荧光标记的反义PNA转染肿瘤细胞后,细胞平均荧光强度显著增强,且呈浓度依赖性。两反义PNA均使SK N SH细胞P gp表达明显降低,阿霉素和长春新碱对细胞的半数抑制浓度值明显下降,MDR 1mRNA表达轻度降低。而PNA1转染对照组C6 /adr细胞后,上述变化不明显。结论 PNA DNA杂交阳离子脂质体转染法可有效增加细胞对PNA的摄取,特异序列的反义PNA可有效逆转神经母细胞瘤的MDR特性。
Objective To investigate the cell transfection method of antisense peptide nucleic acid (PNA) and to reverse the multidrug resistance (MDR) of cultured neuroblastoma in vitro. Methods Two antisense PNA sequences of human MDR 1 gene were designed and synthesized. PNA DNA hybridization and cationic liposome were used to mediate the transfection of SKN SH cell line SK N SH. The transfection efficiency of antisense PNA, the expression of P gp, MDR 1 gene mRNA and the sensitivity to chemotherapeutics were detected by flow cytometry, RT-PCR and MTT respectively. Results Fluorescently labeled antisense PNA transfected tumor cells, the average fluorescence intensity was significantly increased, and in a concentration-dependent manner. Both antisense PNA decreased the expression of P gp in SK N SH cells, while the concentrations of doxorubicin and vincristine significantly decreased the expression of MDR 1 mRNA. The PNA1 transfected control C6 / adr cells, the change was not obvious. Conclusion PNA DNA hybrid liposome transfection method can effectively increase the cellular uptake of PNA, antisense PNA with specific sequence can effectively reverse MDR characteristics of neuroblastoma.