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目的 :探讨NF κB反义寡核苷酸 (oligodeoxynucleotide ,ODN)对人肾小球系膜细胞炎症因子表达的影响 ,为利用NF κB反义ODN治疗肾小球炎性病变奠定基础。 方法 :人工合成p65反义、正义及错配ODN并行全程硫代磷酸化修饰。应用核酸酶保护法观察阳离子脂质体介导的不同浓度的反义ODN(0 0 0 1、0 0 1、0 1、1、1 0μmol/L)对系膜细胞TNF α、IL 1α、IL 1 β、MCP 1、IL 8、TGF β1mRNA表达的影响 ,以正义ODN(1 0 μmol/L)及错配ODN(1 0 μmol/L)作为对照组。 结果 :正常培养状态下 ,系膜细胞可组成型表达TNF α、IL 1 β、IL 8和TGF β1mRNA ,而不表达IL 1α和MCP 1mRNA。细菌脂多糖 (lipopolysaccharide,LPS)刺激后上述 6种炎症因子表达显著上调。p65反义ODN可呈剂量依赖性地抑制LPS诱导的系膜细胞炎性细胞因子TNF α ,IL α,IL 1 β,MCP 1和IL 8的基因表达 ,而对TGF β1无显著抑制作用 ;p65正义及错配ODN均不能抑制炎性细胞因子的表达。 结论 :p65反义ODN可明显抑制LPS诱导的肾小球系膜细胞炎性细胞因子的表达 ,提示NF κB在肾小球疾病进展中起关键性调控作用 ,其反义ODN有可能应用于肾脏病变的实验性治疗之中
OBJECTIVE: To investigate the effect of ODN on the expression of inflammatory cytokines in human glomerular mesangial cells, and to lay the foundation for the treatment of glomerular inflammatory lesions with NF κB antisense oligodeoxynucleotides. METHODS: Synthetic p65 antisense, sense and mismatch ODNs were co-phased with full thiophosphorylation. The effect of cationic liposome-mediated antisense ODN (0 0 0 1, 0 1, 0, 1, 1, 0 μmol / L) on TNFα, IL 1α, IL 1β, MCP 1, IL 8 and TGFβ1mRNA were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The ODN (10 μmol / L) and mismatch ODN (10 μmol / L) Results: Under normal culture conditions, mesangial cells could constitutively express TNFα, IL 1 β, IL 8 and TGF β 1 mRNA but not IL 1 α and MCP 1 mRNA. The expression of six inflammatory cytokines was significantly up-regulated after lipopolysaccharide (LPS) stimulation. The p65 antisense ODN could inhibit the gene expression of inflammatory cytokines TNFα, ILα, IL 1β, MCP 1 and IL 8 induced by LPS in a dose-dependent manner, but had no significant inhibitory effect on TGFβ1; p65 Neither normal nor mismatched ODN inhibited the expression of inflammatory cytokines. CONCLUSION: Antisense ODN of p65 can significantly inhibit the expression of inflammatory cytokines in glomerular mesangial cells induced by LPS, suggesting that NF-κB plays a key regulatory role in the progression of glomerular diseases. Antisense ODN may be applied to the kidney Experimental treatment of lesions