反义肽核酸逆转人神经母细胞瘤多药耐药性的实验研究

Reversion of multidrug resistance of human neuroblastoma by antisense peptide nucleic acid

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 efficiency of a peptide nucleic acid (PNA) delivery system by using liposome via PNA-DNA hybrids. And to test the effectiveness of antisense PNA in reversing multidrug resistance (MDR) in human neuroblastoma cell line SK-N-SH. Methods Two antisense PNAs were designed targeting at the MDR-1 mRNA AUG initiation region and 5′-UTR region. Two PNAs were combined with partially complement DNAs respectively and then delivered into cells using cationic liposome. The transfection efficiency, change of sensitivity to chemotherapy drugs, expression of P-glycoprotein (P-gp) and MDR-1 mRNA were measured by flow cytometry, MTT and RT-PCR respectively. Results Transfection of PNA increased the cell average fluorescence intensity significantly and the extent of increase was dependent on the concentration of PNA. After transfection by both PNAs, P-gp expression and IC50 to ADM and VCR of SK-N-SH cells decreased significantly. But for C6/adr cells as control, PNA1 did not cause such changes. There was slight decrease of the level of MDR-1 mRNA expression after transfection of SK-N-SH cells by both PNAs, but not significant. Conclusions PNA can be delivered into tumor cells in form of PNA-DNA hybrids by cationic liposome. Properly designed antisense PNA can reverse the MDR of SK-N-SH cells efficiently and specifically.