NF-κB靶向性哑铃形“圈套”寡核苷酸体外抑制细胞因子表达效应的研究  

Inhibitory effect of circular dumbbell decoy ODNs targeting NF-кB on cytokines expression in vitro

作　　者：张建国[1] 王惠民[1] 国华[2] 郝天智[2] 杨文军[2] 

机构地区：[1]第三军医大学附属大坪医院野战外科研究所肾内科,重庆400042 [2]第三军医大学附属大坪医院野战外科研究所第一研究室,重庆400042

出　　处：《中国现代医学杂志》2005年第11期1676-1680,共5页China Journal of Modern Medicine

基　　金：重庆市科委应用基础项目(41A11537)

摘　　要：目的设计合成靶向NF-κB的哑铃形“圈套”ODNs,并检测其对NF-κB转录活性和肾小球系膜细胞细胞因子(TNF-α和IL-6)表达的抑制效应。方法采用电泳迁移率改变实验(EMSA)体外检测哑铃形圈套ODNs对NF-κB转录活性的抑制效应。提取大鼠肾小球系膜细胞,随机分为正常对照组、LPS刺激组、哑铃形圈套ODNs处理组、无关圈套ODNs处理组和脂质体处理组。采用阳离子脂质体将2、4和8mg/L不同剂量哑铃形圈套ODNs转染大鼠肾小球系膜细胞,6h后用LPS刺激,收集转染后8、12和18上清和细胞。用酶联免疫吸附试验(ELISA)法检测上清细胞因子蛋白表达,逆转录PCR(RT-PCR)法检测细胞因子mRNA转录。结果哑铃形圈套ODNs在体外能有效地抑制NF-κB与其顺式元件的结合;4、8mg/L剂量哑铃形圈套ODNs可明显抑制LPS诱导的大鼠肾小球系膜细胞TNF-α和IL-6转录与表达。结论靶向NF-κB的哑铃形圈套ODNs在体外可抑制NF-κB的转录活性,从而对体外培养的肾小球系膜细胞细胞因子的表达具有抑制效应。[Objective] To investigate the inhibitory effects of circular dumbbell decoy oligodeoxynucleotides(ODNs) targeting NF-κB on the transcriptional activity of NF-κB binding its cis-elements and the expression of cytokines(TNF-α and IL-6) in mesangial cells in vitro. [Methods] The inhibition effect of Circular dumbbell decoy ODNs on the transcriptional ativity of NF-κB was detected by electrophoretic mobilitory shift assay(EMSA). Mesangial cells extracted from rats were randomly divided into normal control, LPS-stimulated, circular dumbbell decoy-ODNs treated, scrambled-decoy-ODNs treated and cationic liposome treated group. Circular dumbbell decoy ODNs at the doses of 2 mg/L, 4 mg/L and 8 mg/L were transfected respectively into mesangial cells from rats using cationic liposome vector. Mesangial cell were stimulated with LPS at 6 h after circular dumbbell decoy-ODNs transfection. Then,the transfected Supernatants and cells were collected at 8 h, 12 h. cytokines mRNA expression in mesangial cells was detected by RT-PCR and cytokines protein level by ELISA. [Results] Circular dumbbell decoy ODNs could effectively inhibit the DNA binding activity of NF-кB to its cis-elements in vitro. Circular dumbbell decoy ODNs at the doses of 4 and 8 mg/L could effectively inhibit the expression levels of TNF-α, IL-6 mRNA and protein in mesangial cells. [Conclusion] Circular dumbbell decoy ODNs targeting NF-κB can effectively inhibit the expression of cytokines in mesangial cells through the inhibition of NF-κB activity.

关 键 词：NF—κB 哑铃形圈套ODNs 细胞因子 慢性肾小球肾炎 

分 类 号：R692.31[医药卫生—泌尿科学]