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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 construct CRE-transcription factor decoy oligodeoxynucleotide (CRE) in vitro by targeting cAMP response element-binding protein (CREB) -decoy ODN) were used to observe the effects of chronic morphine on the cAMP content, phosphorylated CREB-1 and CREB-1 expression in the cells of human neuroblastoma cell line (SK-N-SH) Provide experimental evidence for finding opioid-dependent target of intervention at the molecular level. METHODS: CRE-decoy ODN was induced in vitro by CRE-decoy ODN with DOTAP (1,2-dioleoyl-3-trimethylammonium propane) as a mediator. Chronic morphine treatment and withdrawal of naloxone induced SK-N-SH cells Co-incubation and electrophoretic mobility shift assay (EMSA) to detect the sequence specificity of CRE-decoy ODN binding to the transcription factor CREB. The uptake of CRE-decoy ODN cells was detected by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Radioimmunoassay (RIA) was used to detect the content of cAMP. Western blot was used to detect the expression of CREB-1 and phosphorylated CREB-1. Results: The cAMP level in the morphine group was significantly higher than that in the physiological sodium chloride solution group (P <0.05), but there was no significant difference between the CRE-decoy ODN group and the physiological sodium chloride solution group (P> 0.05) (P <0.05). Compared with the control group, the expression of phosphorylated CREB-1 in morphine and naloxone group was significantly increased (P <0.01), and the morphine + naloxone group Compared with the control group, CRE-decoy ODN could inhibit the expression of phosphorylated CREB-1 in morphine + naloxone group (P <0.05) ). CONCLUSION: CRE-decoy ODN can inhibit the effects of chronic morphine and naloxone induced withdrawal of cAMP and phosphorylated CREB in SK-N-SH cells.