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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 design a dumbbell-shaped “trap” ODNs targeting NF-κB and to examine its inhibitory effect on the transcriptional activity of NF-κB and the expression of cytokines (TNF-α and IL-6) in mesangial cells. Methods: The NF-κB cis-acting element κB sequence was used as a template to design a circular ODNS containing two copies of κB sequence and a length of 58 bases. The synthesized sequence was then subjected to FAM labeling. The inhibitory effect of dumbbell-shaped ODNs on the NF-κB transcriptional activity was examined by electrophoretic mobility shift assay (EMSA) in vitro. The rat glomerular mesangial cells were extracted and randomly divided into normal control group, LPS stimulation group, dumbbell-shaped ODNs group, ODNs-free group and liposome-treated group. ODNs of 2,4,8 mg / L dumbbell-shaped traps were transfected into rat glomerular mesangial cells with cationic liposomes, and stimulated with LPS after 6 h. The supernatant and cells of transfected cells were collected. The expression of cytokines was detected by enzyme-linked immunosorbent assay (ELISA), and the transcription of cytokines was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Cationic liposomes efficiently transferred dumbbell-shaped ODNs into glomerular mesangial cells. Dumbbell-shaped ODNs could effectively inhibit the binding of NF-κB and its cis-elements in vitro. 4,8mg / L dose dumbbell ODNs could significantly inhibit the transcription and expression of TNF-α and IL-6 in LPS-induced rat mesangial cells. CONCLUSION: The dumbbell-shaped ODNs targeting NF-κB can inhibit the transcriptional activity of NF-κB in vitro and thus have an inhibitory effect on the expression of cytokines in cultured mesangial cells.