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《Journal of The Fourth Military Medical University》 2005-06
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Influence on pertinent index of CREB by CRE decoy oligodeoxynucleotide in SK-N-SH cell line treated by chronic morphine

ZHANG Guo-Zhong,MA Chun-Ling,SU Yan-Jun,ZHANG Jin,ZUO Min,GU Zhen-Yong,YAO Yu-Xia,LI Shu-Jin,CONG BinDepartment of Forensic Medicine,Hebei Medical University, Shijiazhuang 050017,China  
AIM: To investigate the influence on pertinent index of cAMP response element binding protein (CREB) by cAMP response element (CRE) decoy oligodeoxynucleotide in SK-N-SH cell line treated by chronic morphine.METHODS: A single-stranded phosphorothioate oligodeoxynucleotide,composed of the CRE sequence and control oligonucleotides,was synthesized.DOTAP liposomal was used as transfection reagent and the CRE decoy ODN and control oligonucleotides were separately added to SK-N-SH cells culture.In one group,the cells had been treated by chronic morphine and in another group the morphine treated cells had been precipitated by naloxone.The stability of cell-incorporated 32 P-labeled CRE-decoy ODN was examined by nondenaturing polyacrylamide gel electrophoresis and autoradiography.The effect of CRE-decoy ODN on CREB DNA-binding activity was detected by electrophoresis mobility shift assay(EMSA).The proteins of CREB-1 and phosphorylated CREB-1 were detected by western blot.RESULTS: CRE-decoy ODN was successfully transfected to the cells.Compared with that in control groups,chronic morphine and acute naloxone precipitation significantly activated the CREB DNA-binding activity.Phosphorylated CREB-1 increased (P0.01),but the protein of CREB-1 had no obvious change.CRE-decoy ODN specifically downregulated the upper changes(P0.01).CONCLUSION: CRE-decoy ODN can dramatically downregulate the CREB DNA binding activity and phosphorylated CREB-1 induced by chronic morphine and acute naloxone in SK-N-SH cells.
【CateGory Index】: D919
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