反义寡聚脱氧核苷酸抑制脑胶质瘤多药耐药基因1的表达  

Inhibition of Multidrug Resistance Gene 1 Expression in Glioma by Antisense Oligodeoxynucleotides

作　　者：孔建新[1] 宋千[1] 刘延鹏[1] 王成伟[1] 张庆林[1] 

机构地区：[1]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2期211-215,共5页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(30070272)~~

摘　　要：目的研究多药耐药基因1(M D R1)反义寡聚脱氧核苷酸对胶质瘤耐药细胞系C6/adr中M D R1表达的抑制作用。方法以胶质瘤细胞株C6/adr作为胶质瘤体外耐药模型,针对M D R1基因m R NA的A UG起始区-9～+6序列合成反义和相应的正义寡聚脱氧核苷酸,均经硫代磷酸化修饰。反义寡聚脱氧核苷酸(M D R1-AS)序列为5'-CTCCATCA CCAC CTC-3',正义寡聚脱氧核苷酸(M D R1-S)序列为5'-G AG GTG G TG ATG G AG-3'。在脂质体介导下M DR1-A S、M D R1-S转染C6/adr细胞,48h后利用形态学观察和噻唑蓝快速比色法检测M D R1-AS的细胞毒性作用;半定量R T-PCR检测M DR1-A S处理前后的M DR1m RN A的差异表达;流式细胞术分析P糖蛋白(P-gp)的阳性细胞率。结果M D R1-AS对C6/adr细胞无毒性作用;M D R1-AS处理后M D R1m RN A的差异表达由处理前的106%降至30.44%;P-gp阳性表达率亦由处理前的100%降至32.77%。结论M D R1反义寡聚脱氧核苷酸可有效抑制M D R1m R N A及P-gp的表达,从而为基因治疗耐药胶质瘤提供了实验依据,有望开发成为逆转胶质瘤耐药的反义药物。Objective To investigate the effectiveness of phosphorothioate multidrug resistance gene 1(MDR1)antisense oligodeoxynucleotides(MDR1-AS)suppressing MDR1 expression in multidrug-resistant glioma cell line C6/adr. Methods The glioma cell line C6/adr served as the tested model in vitro, MDR1-AS(5'-CTCCATCACCACCTC-3'), complementary to the -9 - +6 sequence of first exon, was syn-thesized and phosphorothioate-modified. As control of sequence specificity, MDR1-S(5'-GAGGTGGTGATGGAG-3')was used. Both antisense and sense oligodeoxynucleotides were transduced to C6/adr cells by lipofectin. The cytotoxity of MDR1-AS was tested using morphological observation and 3-(4,5-dime-thylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Semi-quantitative reverse transcription polymerase chain reaction was used to monitor the expression levels of the MDR1 mRNA in the different groups. The positive rate of the MDR1 gene product P glycoprotein(P-gp)was determined by flow cytometry assessment. Results No cytotoxicity of MDR1-AS was observed. The MDR1 mRNA expression level was decreased from 106% to about 30.44% 48 h after MDR1-AS treatment. The P-gp positive rate of MDR1-AS treated C6/adr cells decreased from 100% to 32.77%, with that of C6/adr cells considered as 100%. Conclusions MDR1-AS can effectively inhibit MDR1 expression in the C6/adr cell line at both the mRNA and protein level, and may be an alternative treatment of drug-resistant gliomas.

关 键 词：胶质瘤 反义寡聚脱氧核苷酸 多药耐药 

分 类 号：R739.41[医药卫生—肿瘤]