CRE-decoy ODN对慢性吗啡作用的SK-N-SH细胞中相关信号分子的影响  

Effects of CRE-decoy ODN on the signal molecules after chronic morphine treatment in SK-N-SH cells

作　　者：段立华[1] 张瑾[2] 

机构地区：[1]河北化工医药职业技术学院,石家庄050026 [2]石家庄市第二人民医院,石家庄050000

出　　处：《中国新药杂志》2009年第11期1032-1037,共6页Chinese Journal of New Drugs

摘　　要：目的:以环磷酸腺苷(cAMP)反应元件结合蛋白(Cyclic-AMP response-element-binding protein,CREB)为靶点,体外合成CRE转录因子诱骗寡核苷酸(CRE-transcription factor decoy oligodeoxynucleotide,CRE-decoy ODN),观察其对慢性吗啡作用和纳络酮催促戒断的人神经母细胞瘤细胞株(SK-N-SH)细胞cAMP含量、磷酸化CREB-1及CREB-1表达的影响,为在分子水平上寻找阿片类依赖的干预靶点提供实验依据。方法:体外合成CRE-decoy ODN,以DOTAP(1,2-dioleoyl-3-trimethylammonium propane)为转移介质,将CRE-decoy ODN与慢性吗啡作用和纳络酮催促戒断的SK-N-SH细胞共同孵育,电泳迁移率改变分析(EMSA)检测CRE-decoy ODN与转录因子CREB结合的序列特异性;采用非变性聚丙烯酰胺凝胶电泳及放射自显影检测CRE-decoy ODN细胞摄取;放射免疫分析法(RIA)检测细胞cAMP含量;Western blot检测CREB-1及磷酸化CREB-1表达。结果:单纯吗啡组较生理氯化钠溶液对照组细胞cAMP水平显著升高(P<0.05),单纯CRE-decoy ODN组与生理氯化钠溶液对照组无明显差异(P>0.05),但能明显抑制吗啡及纳络酮组的cAMP水平(P<0.05);单纯吗啡组与生理氯化钠溶液对照组比较,磷酸化CREB-1表达明显增高(P<0.01),吗啡+纳络酮组较单纯吗啡组轻度降低,与对照组比较仍有显著差异(P<0.01);CRE-decoyODN可抑制单纯吗啡组及吗啡+纳络酮组磷酸化CREB-1表达明显增高(P均<0.05)。结论:CRE-decoyODN可抑制慢性吗啡作用及纳络酮催促戒断引起SK-N-SH细胞cAMP和磷酸化CREB表达的升高。Objective： To observe the effect of CRE-transcription factor decoy oligodeoxynucleotide（CRE- decoy ODN） on the content of cAMP, cyclic AMP response-element-binding protein-1 （CREB-1） and phosphorylated CREB-1 in SK-N-SH cells after chronic morphine treatment. Methods： A single-stranded phosphorothioate oli- godeoxynucleotide composed of the CRE sequence and control oligonucleotides were synthesized in vitro. The CRE- decoy ODN and control oligonucleotides were separately introduced to SK-N-SH cells by DOTAP（ 1,2-dioleoyl-3-trimethylammonium propane） liposome, which had been treated by chronic morphine or precipitated by naloxone. The stability of cell-incorporated 32P-labeled CRE-decoy ODN was detected by nondenaturing polyacrylamide gel electrophoresis and autoradiography. The effect of CRE-decoy ODN on the DNA-binding activity of extract of Hela nucleolus was detected by electrophoresis mobility shift assay（EMSA）. The content of cAMP was measured by radioimmunoassay （RIA） ; CREB-1 and phosphorylated CREB-1 were detected by Western blot. Results： The cAMP content was increased in the cells after chronic morphine treatment（P 〈 0.05 ） as compared with the control cells. Treatment with CRE-decoy ODN obviously decreased the cAMP content in morphine-treated and acute naloxoneprecipitated cells as compared with the control cells（P 〈0.05）. Phosphorylated CREB-1 in morphine-treated and acute naloxone-precipitated cells was markedly increased as compared with control cells（P 〈 0.01 ） ; this increase was obviously inhibited by treatment with CRE-decoy ODN （P 〈 0.05）. Conclusions： CRE-decoy ODN inhibits the increased contents of cAMP and phosphorylated CREB-1 induced by chronic morphine treatment and acute naloxone-precipitation in SK-N-SH cells.

关 键 词：吗啡 SK—N—SH细胞株 CRE转录因子诱骗寡核苷酸(CRE—decoy ODN)CRE—decoy ODN 环 磷酸腺苷(cAMP) cAMP反应元件结合蛋白-1(CREB-1) 磷酸化CREB-1 

分 类 号：R965.1[医药卫生—药理学] R964[医药卫生—药学]