NF-κB靶向性哑铃形“圈套”寡核苷酸对体外肾小球系膜细胞细胞因子表达的影响  

Inhibitory effect of circular dumbbell decoy ODNs targeting NF- кB on cytokinesexpression in mesangial cells 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,重庆市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,重庆市400042

出　　处：《实用医学杂志》2005年第12期1257-1260,共4页The Journal of Practical Medicine

基　　金：重庆市科委应用基础项目(编号: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: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ODNS,合成后部分序列做FAM标记,行转集效率鉴定实验。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、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、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:阳离子脂质体可将哑铃形圈套ODNs高效转集入肾小球系膜细胞,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 dum bb ell decoy oligod eoxynucleotides(ODNs )targeting NF-κB on the transcriptiona l activity of N F-κB binding its cis-elements and the ex pression of cytokine s(TNF-αand IL-6)in mesangial cells in vitro.Methods According toκB sequ ence,circular dumbbell decoy-ONDs including 58bases and 2copies ofκB seque nces were designed and synthesized.The inhibition effect of circular dumbbell d ecoy ODNs on th e transcriptional activity of NF-κB was detected by electropho retic mobilitory shift assay(EMSA).Mesangial cells extracted from rats were r andomly divided into normal c ontrol,LPS-stimulated,circular dumbbell decoy- ODNs treated,scram bled-decoy-ODNs treated and cation ic liposome treated gro up.Circular dumbbell decoy ODNs at the doses of 2mg /L,4mg /L and 8mg /L were transfected respectively into mesangial c ells from rats using cationic li poso me vector.Mesangial cell were stimulated with LPS at 6h after c ircular d umbbell decoy-ODNs transfection.Then,the transfected Supernatants and cells w ere collected at 8h,12h.Cytokines mRNA expressio n in mesangial cells was d etected by RT-PCR and cytokines protein level by ELISA.Results Circular dumbbe ll decoy ODNs could e ffectively inhibit the DNA binding a ctivity of NF-кB to its cis-elements in vitro.Circular dumbbell decoy ODNs at the doses of 4and 8mg /L could e ffectively inhibit the expression l evels of TNF-α,IL-6mRN A and protein in mesangial c ells.Conclusion Circular dumbbell decoy ODNs targe ting NF-κB could effectively inhibit the expr ession of cytokines in mesangial cells through the inhibition of NF-κB activity.[

关 键 词：肾小球系膜细胞 NF-kB 哑铃形 细胞因子表达 酶联免疫吸附试验(ELISA) 寡核苷酸 圈套ODNs 靶向性 阳离子脂质体 TNF-α 抑制效应 转录活性 顺式作用元件 MRNA转录 电泳迁移率 正常对照组 LPS诱导 设计合成 ODNS 体外检测 

分 类 号：R692.3[医药卫生—泌尿科学]