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 (IL - 6和 TGF-β1)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、L PS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、8mg/ L 不同剂量哑铃形圈套 ODNs转染大鼠肾小球系膜细胞 ,6 h后用 L PS刺激 ,收集转染后 8、12、18上清和细胞。用酶联免疫吸附试验 (EL ISA)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 m RNA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 L PS诱导的大鼠肾小球系膜细胞 IL - 6和 TGF-β1 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 。
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(IL-6 and TGF-β_1) in mesangial cells. Methods The inhibitory effect of circular dumbbell decoy ODNs on the transcriptional activity 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 IL-6, TGF-β1 mRNA and protein in mesangial cells.Conclusion Circular dumbbell decoy ODNs targeting NF-κB could effectively inhibit the expression of cytokines in mesangial cells through the inhibition of NF-κB activity.
出处
《西部医学》
2004年第4期289-292,共4页
Medical Journal of West China
基金
重庆市科委应用基础项目 (41 A1 1 537)
关键词
NF—κB
哑铃形圈套ODNs
细胞因子
Nuclear factor kappa B
Circular dumbbell decoy oligodeoxynucleotides
Cytokines
