摘要
目的:研究以转录因子cAMP反应元件结合蛋白(CREB)为靶点的CRE -decoyODN对慢性吗啡诱导SK -N -SH细胞胆囊收缩素(CCK)及fosBmRNA表达上调的抑制作用。方法:体外合成含cAMP反应元件CRE序列TGACGTCA的单链寡核苷酸,将自身杂交形成发卡结构。将终浓度为15 0nmol/L的CRE -decoyODN与SK -N-SH细胞孵育1h后,加入终浓度为10 0 μmol/L的吗啡作用4 8h ,随后加入终浓度为10 μmol/L的纳络酮,戒断15min。采用电泳迁移率改变分析(EMSA)检测CRE -decoyODN与CREB结合的序列特异性及其对慢性吗啡诱导的CREB的DNA结合活性升高的影响;细胞掺入的CRE -decoyODN用酚:氯仿法提取,经2 0 %非变性聚丙烯酰胺凝胶电泳及放射自显影检测;采用RT -PCR检测CCK及fosBmRNA表达。结果:慢性吗啡作用及纳络酮急性戒断使SK-N -SH细胞CREB的DNA结合活性、CCK和fosBmRNA表达明显升高,CRE -decoyODN可特异抑制其升高。结论:CRE -decoyODN通过特异抑制慢性吗啡诱导SK -N -SH细胞CREB的DNA结合活性而下调CCK及fosBmRNA表达。
AIM: To investigate the inhibitory effect s of a synthetic CRE-transcription factor decoy oligodeoxynucleotide (CRE-decoy ODN) on the upregulation of the expression of cholecystokinin (CCK) and fosB mRN A induced by chronic morphine administration in SK-N-SH cells. METHODS: The CRE cis-element, TGACGTCA, was palindromic, a sy nthetic single-stranded phosphorothioate oligodeoxynucleotide composed of the CR E sequence self-hybridizes to form a duplex/hairpin. The CRE-palindromic decoy a nd control oligodeoxynucleotides were added to the medium (1 h before exposure t o morphine) at 150 nmol/L in the presence of cationic lipid DOTAP. After the cel ls were treated with 100 μmol/L morphine for 48 h, 10 μmol/L naloxone was use d for 15 min. The effects of CRE-decoy ODN on the DNA-binding activity of CREB, the expression of CCK and fosB mRNA were detected by electrophoresis mobi lity shift assay (EMSA) and RT-PCR, respectively. The stability of cell-incorpo rated [ 32P]-labeled CRE-decoy ODN was extracted with phenol:chloroform a nd then subjected to 20% nondenaturing polyacrylamide gel electrophoresis and au toradiography. RESULTS: Chronic morphine administration and acute naloxone-prec ipitated withdrawal significantly activated the DNA-binding activity of CREB and the expression of CCK and fosB mRNA in SK-N-SH cells. The CRE-decoy ODN pen etrated into the cells, specifically downregulated these indexes. CONCLUSIONS: CRE-decoy ODN can significantly downregulates the e xpre ssion of CCK and fosB mRNA through specifically suppressing the DNA-binding activity of CREB activated by chronic morphine administration in SK-N-SH cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2005年第4期630-635,共6页
Chinese Journal of Pathophysiology
