【摘要】 目的研究转录因子CREB的结合位点cAMP反应元件(cAMPresponseelement,CRE)的诱骗寡核苷酸(CREtranscriptionfactordecoyoligodeoxynucleotide,CREdecoyODN)对吗啡依赖SKNSH细胞的神经元型一氧化氮合酶(Neuronalnitricoxidesynthase,nNOS)及fosBmRNA表达上调的抑制作用。方法建立吗啡依赖SKNSH细胞模型,体外合成含CRE序列的寡核苷酸,作为CREdecoyODN,与阳离子脂类N[1(2,3dioleoyloxy)propyl]N,N,Ntrimethylammoniummethylsulfate(DOTAP)混合后导入细胞。采用放射自显影检测细胞内参入的CREdecoyODN。采用电泳迁移率改变分析(Electrophoresismobilityshiftassay,EMSA)及RTPCR技术,分别检测CREdecoyODN对吗啡依赖诱导的CREB的DNA结合活性、nNOS及fosBmRNA表达的影响。结果CREdecoyODN可在细胞内稳定存在,特异抑制吗啡依赖SKNSH细胞CREB的DNA结合活性升高、nNOS和fosBmRNA表达上调。结论CREdecoyODN通过特异抑制吗啡依赖SKNSH细胞的CREB的DNA结合活性而下调nNOS及fosBmRNA表达。更多还原

【Abstract】 Aim To investigate the inhibitory effects of a synthetic CRE-transcription factor decoy oligodeoxynucleotide (CRE-decoy ODN) on the upregulation of the mRNA expression of nNOS and fosB in morphine-dependent SK-N-SH cells. Methods Morphine-dependent SK-N-SH cells were used. A synthetic single-stranded phosphorothioate oligodeoxynucleotide composed of the cAMP response element (CRE) sequence used as the CRE-decoy ODN in the presence of cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) was added to the culture medium. The effects of the CRE-decoy ODN on the DNA-binding activity of CREB, the expression of neuronal nitric oxide synthase (nNOS) and fosB mRNA were detected by electrophoresis mobility shift assay (EMSA) and RT-PCR respectively. Results Morphine dependence significantly activated the DNA-binding activity of CREB and the expression of nNOS and fosB mRNA. The CRE-decoy ODN could penetrate into SK-N-SH cells, specifically and stably suppress these indexes. Conclusion The CRE-decoy ODN can downregulate the expression of nNOS and fosB mRNA through specifically suppressing the DNA-binding activity of CREB in morphine-dependent SK-N-SH cells.更多还原