反义肽核酸阻断人神经母细胞瘤多药耐药相关蛋白P-糖蛋白的表达  

Inhibition of Multidrug Resistance Related P-gp Expression in Human Neuroblastoma by Antisense Peptide Nucleic Acid

作　　者：郭华 张庆林[2] 张健 王成伟[2] 孔建新[2] 刘福生[2] 马道新[2] 卞继峰[2] 

机构地区：[1]Department of Neurosurgery,Shandong Provincial Hospital,Jinan [2]山东大学第二医院神经外科,济南250033

出　　处：《中国医学科学院学报》2005年第3期300-304,共5页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(30070272)~

摘　　要：目的探讨反义肽核酸(PNA)的细胞转染方法及其对体外培养的神经母细胞瘤多药耐药(MDR)相关蛋白P-糖蛋白(P-gp)表达的影响。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。采用流式细胞术、逆转录聚合酶链反应(RT-PCR)和高效液相色谱等方法分别检测反义PNA的转染效率,转染前后细胞P-gp、MDR-1基因mRNA的表达及细胞内阿霉素(ADM)浓度。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,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,MDR-1mRNA表达轻度降低,细胞内ADM聚集浓度明显增加。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可在一定程度上阻断神经母细胞瘤MDR相关蛋白P-gp的表达。Objective To investigate the efficiency of a peptide nucleic acid(PNA)delivery system by using liposome via PNA-DNA hybrids and to test the inhibitive action of antisense PNA on expression of multidrug resistance(MDR)related P-glycoprotein(P-gp)in human neuroblastoma cell line SK-N-SH. Methods Two antisense PNAs were designed targeting at MDR-1 mRNA and then combined with partially complement DNAs respectively. The hybrids were delivered into cells using cationic liposome. The transfection efficiency, expression of P-gp and MDR-1 mRNA, intracellular adarimycin(ADM)were measured by flow cytometry, reverse transcription-polymerase chain reaction(RT-PCR), and high performance liquid chromatography(HPLC). Results Transfection of PNA increased the cell average fluorescence intensity significantly and the extent of increase was dependent on the concentration of PNA. After being transfected by both PNAs, P-gp expression of SK-N-SH cells decreased significantly and the intracellular ADM level was increased by about 3 times. The level of MDR-1 mRNA expression slightly decreased after transfection, but no statistical significance was observed. Conclusions PNA can be delivered into tumor cells in form of PNA-DNA hybrids by cationic liposome. Properly designed antisense PNA can inhibit MDR related P-gp expression of SK-N-SH cells efficien- tly and specifically.

关 键 词：神经母细胞瘤 多药耐药性 肽核酸 反义 

分 类 号：R73-36[医药卫生—肿瘤] 2[医药卫生—临床医学]