本文選題：嗎啡依賴 + SK-N-SH細(xì)胞　； 參考：《河北醫(yī)科大學(xué)》2005年博士論文

【摘要】：第一部分 CRE-decoy ODN對嗎啡依賴SK-N-SH細(xì)胞CREB的DNA結(jié)合活性升高的抑制作用 目的:研究以轉(zhuǎn)錄因子cAMP反應(yīng)元件結(jié)合蛋白(cAMP response element binding protein, CREB)為靶點(diǎn)的CREB結(jié)合位點(diǎn)誘騙寡核苷酸(CRE-transcription factor decoy oligodeoxynucleotide, CRE-decoy ODN)對慢性嗎啡誘導(dǎo)SK-N-SH細(xì)胞CREB的DNA結(jié)合活性的影響。 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'。順式元件CRE序列TGACGTCA為回文結(jié)構(gòu),合成含CRE序列的硫代磷酸化修飾的單鏈寡核苷酸,可自身雜交形成發(fā)卡結(jié)構(gòu)。陽離子脂類N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP)為轉(zhuǎn)移介質(zhì)。使用時(shí)分別按ODN:HBS=1:10(μg/μl)、DOTAP:HBS=1:2.3(μg/μl)將ODN與DOTAP稀釋后,再按ODN:DOTAP=1:6(μg/μg)將兩者混勻,于15～25℃放置10～15 min。(2) 細(xì)胞培養(yǎng)和實(shí)驗(yàn)分組:人神經(jīng)母細(xì)胞瘤SK-N-SH細(xì)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培養(yǎng)液中,于37℃含5%CO_2孵育箱中培養(yǎng)。細(xì)胞融合到約50%時(shí),在培養(yǎng)液內(nèi)加入終濃度為100 μmol/L的鹽酸嗎啡,作用48 h,隨即加入終濃度為10 μmol/L的鹽酸納絡(luò)酮,作用15 min。在加入嗎啡前1 h,分別在培養(yǎng)液內(nèi)加入DOTAP、ODN與DOTAP混合物。ODN終濃度為150 nmol/L。實(shí)驗(yàn)分為5組,即①非治療組:包括生理鹽水對照(C)、慢性嗎啡作用(M)及嗎啡+納絡(luò)酮(M+N)亞組;②CRE-decoy ODN治療組:包括單獨(dú)CRE-decoy ODN治療、M+CRE-decoy ODN及M+N+CRE-decoy ODN亞組;③mismatch ODN治療組:包括單獨(dú)mismatch ODN治療、M+mismatch ODN及M+N+mismatch ODN亞組;④nonsense ODN治療組:括單獨(dú)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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