NF-κB靶向性哑铃形"圈套"寡核苷酸对肾小球系膜细胞TNF-α的表达抑制作用的研究

Inhibitory Effect of Circular Dumbbell Decoy ODNs Targeting NF-KB on TNF-α Expression in Mesangial Cells

目的:本实验设计合成靶向NF-κB的哑铃形圈套ODNs,并检测其对肾小球系膜细胞TNF-α表达的抑制效应。方法:以NF-κB顺式作用元件κB序列为模板,设计包含两个拷贝的κB序列,长度为58个碱基的环状IDNs,合成后部分序列做FAM标记,行转染效率鉴定实验。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、8 mg／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、18上清和细胞。用酶联免疫吸附试验(ELISA)法检测上清TNF-α蛋白表达,逆转录PCR(RT-PCR)法检测TNF-αmRNA转录。结果:阳离子脂质体可将哑铃形圈套ODNs高效转染入肾小球系膜细胞;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转录和表达。结论:靶向NF-κB的哑铃形圈套ODNs对体外培养的肾小球系膜细胞炎性因子TNF-α的表达具有抑制效应。

Objective To study the inhibitory effects of circular dumbbell decoy oligodeoxynucleotides targeting NF κB on the expression of TNF-α in mesangial cells. Methods According to κB sequence, circular dumbbell decoy-ODNs including 58 bases and 2 copies of κB sequences were designed and synthesized. Mesangial cells of rats were extracted and randomly divided into normal control, LPS-stimulated, circular dumbbell decoy-ODNs treated, scrambled-decoy-ODNs treated and cationic liposome treated groups. With carriers of cationic liposomes, 2 mg/L,4 mg/L, and 8 mg/L decoy ODNs were respectively transfected into mesangial cells of rats. The mesangial cells were stimulated with LPS 6h after circular dumbbell decoy-ODNs transfection. The supernatants and cells were collected at 8, 12 and 18 hours after decoy-ODNs transfection. TNF-α mRNA transcription in the mesangial cells was assessed by RT-PCR and TNF-αprotein expression was determined by ELISA. Results Circular dumbbell decoy ODNs could effectively inhibit the DNA binding activity of NF -κB in vitro. After using decoy ODNs, the expression levels of TNF-α mRNA and protein in the mesangial cells significantly decreased. Conclusions Circular dumbbell decoy ODNs targeting NF-κB could effectively inhibit the expression of TNF-α in the mesangial cells.