转录因子NF-κB和E2F的decoy寡核苷酸抑制血管平滑肌细胞的增殖  

Inhibitive effect of decoy oligonucleotides of transcription factor NF-κB and E2F on the proliferation of vascular smooth muscle cells

作　　者：席锐[1] 陈桢玥[1] 周俊[1] 吴春芳[1] 陆国平[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院心脏科,上海市200025

出　　处：《中国临床康复》2006年第9期106-108,i0006,共4页Chinese Journal of Clinical Rehabilitation

基　　金：上海市科学技术委员会资助基础研究科研项目(02JC14037)~~

摘　　要：目的:探讨转录因子NF-κB和E2F的decoy寡核苷酸单独或联合应用对猪血管平滑肌细胞增殖的抑制作用及其机制。方法:实验于2004-05/2005-08在瑞金医院内科实验室完成。①原代培养小型猪胸主动脉平滑肌细胞,体外合成转录因子NF-κB和E2F的decoy寡核苷酸,将细胞分为6组:正常对照组、NF-κBdecoy寡核苷酸组、E2Fdecoy寡核苷酸组、NF-κB+E2Fdecoy寡核苷酸组、NF-κB错配寡核苷酸组、E2F错配寡核苷酸组,以脂质体为载体转染细胞,采用激光扫描共聚焦显微术观察FITC标记的寡核苷酸细胞内定位情况。②各组细胞经同步化处理后,予体积分数为0.1的胎牛血清刺激,采用四甲基偶氮唑盐法检测NF-κB和E2F的decoy寡核苷酸对细胞增殖的抑制作用。③免疫印迹法检测增殖细胞核抗原表达水平。④流式细胞仪碘化丙啶法及磷脂结合蛋白V法分析细胞周期及细胞凋亡情况。结果:①FITC标记的寡核苷酸主要聚集于细胞核内,转染效率为(46.67±5.77)%。②体积分数为0.1的胎牛血清刺激24h后,NF-κBdecoy寡核苷酸组、E2Fdecoy寡核苷酸组和NF-κB+E2Fdecoy寡核苷酸组均可明显抑制细胞增殖犤(34.96±6.35)%,(38.75±0.14)%,(49.60±5.07)%犦,并且decoy寡核苷酸组抑制作用普遍强于错配寡核苷酸组,联合应用组作用强于单独应用组(P<0.01)。胎牛血清刺激48h后NF-κBdecoy寡核苷酸组和NF-κB+E2Fdecoy寡核苷酸组仍可明显抑制细胞增殖犤(36.35±5.24)%,(42.60±4.21%)犦,但E2Fdecoy寡核苷酸组增殖反而超过正常对照组犤(-24.22±9.68)%犦(P<0.01)。③胎牛血清刺激24h后NF-κB和E2F的decoy寡核苷酸单独或联合应用组均可降低增殖细胞核抗原的表达。④decoy寡核苷酸使停滞在DNA合成前期的细胞百分数增加,早期凋亡增加。结论:在原代培养的猪的血管平滑肌细胞,NF-κBdecoy寡核苷酸和E2Fdecoy寡核苷酸单独或联合应用可以抑制细胞增殖,但E2Fdecoy寡核苷酸单独应用只�AIM： To probe into the inhibition effect of decoy oligonucleotides（ODNs） of transcription factor NF-κB and /or E2F on the proliferation of the vascular smooth muscle cells of porcine and its mechanism. METHODS： This experiment was conducted at the Laboratory of Internal Medicine, Ruijin Hospital from May 2004 to August 2005. ①We cultured porcine thoracic artery smooth muscle cells and synthesized ODNs in vitro. Cells were divided into 6 groups： normal control group, NF-KB decoy ODNs group, E2F decoy ODNs group, NF-κB＋E2F decoy ODNs group, NF-KB mismatched ODNs group, E2F inismatched ODNs group and transfected by ODNs with liposome. We applied Laser Scanning Confocal microtechnic to observe the localization of ODNs labeled by FTTC. ②Cells Were stimulated by 0.1 fetal bovine serum after synchronized , then methyhhiasolyl tetrazolium assay （MTT）was applied to detect the inhibition effect on proliferation. ③ Western Blotting was used to detect the expression of proliferating cell nuclear antigen （PCNA）. ④We also used flow cytometry and phospholipids heptoglobin V method to analyze the condition of the cell cycle and apoptosis. RESULTS： ① ODNs labled by FITC mainly gathered in the cell nucleus and the transfection rate was （46.67±5.77）%. ② After stimulated by 0.1 fetal bovine serum for 24 hours, the inhibition effect of NF-κB decoy ODNs group, E2F decoy ODNs group and NF-κB＋E2F decoy ODNs group on cellular proliferation was all obvious [（34.96±6.35）%, （38.75±0.14）% , （49.60 ±5.07）% ],and the inhibition effect of decoy ODNs group was all stronger than that of the mismatched ODNs group, and the inhibition effect of combination application group was stronger than that of single application group （P 〈 0.01）. After stimulated by fetal bovine serum for 48 hours, NF-κB decoy ODNs group and NF-κB＋E2F decoy ODNs group still could obviously inhibit cellular proliferation [（36.35 ±5.24）%, （42.60±4.21%）], but the proliferation of E2F decoy ODNs group

关 键 词：肌细胞 平滑肌 寡核苷酸类 转录因子 基因治疗 

分 类 号：R322.1[医药卫生—人体解剖和组织胚胎学]