CREB结合位点诱骗寡核苷酸缓解吗啡依赖机制的实验研究

发布时间：2018-04-13 20:51

  本文选题：吗啡依赖 + SK-N-SH细胞　； 参考：《河北医科大学》2005年博士论文

【摘要】：第一部分 CRE-decoy ODN对吗啡依赖SK-N-SH细胞CREB的DNA结合活性升高的抑制作用 目的:研究以转录因子cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)为靶点的CREB结合位点诱骗寡核苷酸(CRE-transcription factor decoy oligodeoxynucleotide, CRE-decoy ODN)对慢性吗啡诱导SK-N-SH细胞CREB的DNA结合活性的影响。 方法:(1) 合成全硫代磷酸化ODN:CRE-decoy ODN:5'-TGACGTCA TGACGTCA TGACGTCA-3′; CRE mismatch ODN; 5'-TGTGGTCA TGTGGTCA TGTGGTCA-3'; nonsense palindromic ODN; 5'-CTAGCTAG CTAGCTAG CTAGCTAG-3'。顺式元件CRE序列TGACGTCA为回文结构,合成含CRE序列的硫代磷酸化修饰的单链寡核苷酸,可自身杂交形成发卡结构。阳离子脂类N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP)为转移介质。使用时分别按ODN:HBS=1:10(μg/μl)、DOTAP:HBS=1:2.3(μg/μl)将ODN与DOTAP稀释后,再按ODN:DOTAP=1:6(μg/μg)将两者混匀,于15～25℃放置10～15 min。(2) 细胞培养和实验分组:人神经母细胞瘤SK-N-SH细胞株在含0.1 mmol/L非必需氨基酸、10%胎牛血清及2 mmol/L L-谷氨酰胺的MEM培养液中,于37℃含5%CO_2孵育箱中培养。细胞融合到约50%时,在培养液内加入终浓度为100 μmol/L的盐酸吗啡,作用48 h,随即加入终浓度为10 μmol/L的盐酸纳络酮,作用15 min。在加入吗啡前1 h,分别在培养液内加入DOTAP、ODN与DOTAP混合物。ODN终浓度为150 nmol/L。实验分为5组,即①非治疗组:包括生理盐水对照(C)、慢性吗啡作用(M)及吗啡+纳络酮(M+N)亚组;②CRE-decoy ODN治疗组:包括单独CRE-decoy ODN治疗、M+CRE-decoy ODN及M+N+CRE-decoy ODN亚组;③mismatch ODN治疗组:包括单独mismatch ODN治疗、M+mismatch ODN及M+N+mismatch ODN亚组;④nonsense ODN治疗组:括单独nonsense
[Abstract]:The first part of the inhibitory effect of CRE-decoy ODN on the increase of DNA binding activity of CREB in morphine-dependent SK-N-SH cellsAim: to investigate the effect of CREB binding site targeting the transcription factor cAMP response element binding protein camp response element binding (CREBB) on DNA binding activity of CREB induced by chronic morphine in SK-N-SH cells induced by chronic morphine, and the effect of factor factor decoy oligodeoxynucleotiotide (CRE-decoy ODN) on the DNA binding activity of SK-N-SH cells induced by chronic morphine.Methods Total thiophosphorylation of ODN:CRE-decoy ODN:5'-TGACGTCA TGACGTCA TGACGTCA-3N; CRE mismatch ODN; 5'-TGTGGTCA TGTGGTCA TGTGGTCA-3; nonsense palindromic ODN; 5'-CTAGCTAG CTAGCTAG CTAGTAG-3 were synthesized.The cis element CRE sequence TGACGTCA is a palindrome structure, and the thiophosphorylation modified single strand oligonucleotide containing CRE sequence is synthesized, and the hairpin structure can be formed by self hybridization.The cationic lipids N- [1-O2O2-dioleoyloxypropyl] -N-trimethylammonium methylsulfate (DOTAP) were used as the transfer medium.When using, ODN and DOTAP were diluted by ODN: HBS1: 10 (渭 g / 渭 l / 渭 g / 渭 l) respectively, and then mixed by ODN: DOTAP 1: 6 (渭 g / 渭 g).Human neuroblastoma SK-N-SH cell line was cultured in 10% fetal bovine serum containing 0.1 mmol/L non-essential amino acid and 2 mmol/L L-glutamine in MEM medium and incubated in 5%CO_2 incubator at 37 鈩，

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