dsODNs 靶向封闭肺泡巨噬细胞中 NF -κB炎症信号通路的实验研究

An experimental study on targeted blockage of NF-κB inflammatory signaling pathway with dsODNs in alveolar macrophages （AMs）

目的：在确定脂质体转染双链寡聚脱氧核苷酸（ dsODNs ）最佳转染浓度及时间的基础上，评估脂质体转染的双链寡聚脱氧核苷酸（ dsODNs/Lipofectamin2000）竞争性抑制对肺泡巨噬细胞中核因子κB（ NF-κB）/DNA结合活性的影响，验证dsODNs-decoy策略靶向封闭肺泡巨噬细胞中NF-κB信号通路的可行性。方法支气管肺泡灌洗分离提取家兔肺泡巨噬细胞（ AMs）后体外培养，以Lipofectamine2000为载体转染dsODNs后对巨噬细胞进行干预，测定巨噬细胞中dsODNs/Lipofectamine2000的转染活性、转染效率及转染后对AMs的毒性；检测dsODNs/Lipofectamine2000转染对炎症因子（ IL -1α、IL -6、TNF -α等） mRNA 的表达；观察 dsODNs/Lipofectamine2000转染后NF -κB p65亚基的核移位情况。结果转染肺泡巨噬细胞dsODNs/Lipofectamine2000的最适比例为1∶5，最佳时间为6 h，此时细胞毒性适中，转染效率、荧光强度及细胞状态最佳；与LPS组比较，dsODNs/Lipofectamine2000转染组各种炎症介质mRNA的表达均明显抑制（P＜0．01）；细胞核中NF-κB p65亚基的表达明显少于细胞浆中。结论 dsODNs/Lipofecetamine2000能在体外有效转染AMs并成功抑制AMs中NF-κB信号通路相关炎症靶基因的转录表达，证实了dsODNs-decoy策略影响炎症效应细胞的可行性。

Objective After determined the optimal transfection concentration and time , evaluate the effectiveness of the inhibition of （ NF-κB）/DNA binding activity by lipofectamine 2000 transfected double -stranded oligodeoxynucleotides （ dsODNs ） in order to test the feasibility of dsODNs-decoy strategy targeted on NF -κB pathway in alveolar macrophages （ AMs ） .Methods AMs were separated from bronchoalveolar lavage and cultured for dsODNs transfection by Lipofectamine2000.The effectiveness of dsODNs/Lipofectamine2000 transfection activity, the optimum concentration ratio and transfection rate in AMs were investigated . Transfection toxicity of Lipofectamine2000 was detected using LDH kits .The expression levels of IL -1α, IL-6, TNF -αmRNA were determined by RT -PCR.Nuclear translocation of p65 after dsODNs/Lipofectamine2000 transfected in AMs was detected by Western -blot.Resu lts The transfection rate was best and cytotoxicity was moderate when the concentration ratio of dsODNs /Lipofectamine2000 was 1∶5.The cell transfection efficiency , fluorescence intensity and cells activity were best at the time spot of 6 hours after transfection.IL-1α, IL-6 and TNF-αmRNA expression were all decreased in the treatment group compared with LPS group （P 〈0.01）.NF -κB p65 translocation to the nuclei was significantly blocked.Conclusion Lipofectamine 2000 is effective in the transfection of dsODNs into AMs and＆amp;nbsp;dsODNs-decoy can inhibit the gene expression by blocking the NF -κB pathway.It is viable to use dsODNs-decoy strategy to affect inflammatory in AMs .