线粒体DNA诱导肺泡巨噬细胞炎症反应的实验研究

Induction of Alveolar Macrophage Inflammatory Response by Mitochondrial DNA

目的观察线粒体DNA(mitochondrial DNA,mtDNA)刺激肺泡巨噬细胞后炎症因子的动态变化,探讨mtDNA对肺泡巨噬细胞的致炎效应。方法取SD大鼠肝脏组织用改良的碱变性法进行mtDNA的提取、纯化。将体外培养的大鼠肺泡巨噬细胞(NR8383)分为mtDNA组(5μg·mL-1,A组),mtDNA组(10μg·mL-1,B组),空白对照组(C组),分别在A、B组中加入mtDNA 5μg·mL-1、10μg·mL-1,在0、3、6、12和24 h时相点收集各组细胞培养上清液,采用酶联免疫吸附法(ELISA)检测细胞培养上清液中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和IL-6的表达水平。结果 A组和B组在3、6、12和24 h TNF-α、IL-1β和IL-6的表达量显著高于C组(P<0.01),且在3、6、12和24 h时B组的表达量高于A组(P<0.05)。结论 mtDNA能够诱导肺泡巨噬细胞TNF-α、IL-1β和IL-6等炎症因子的表达,且呈剂量依赖性,推测mtDNA具有促炎作用。

Obj ective To observe the dynamic changes in inflammatory cytokines in alveolar macro-phages stimulated by mitochondrial DNA（mtDNA）,and to explore the inflammatory effect of mtDNA on alveolar macrophages.Methods The mtDNA was extracted and purified from SD rat liver tissue using a modified alkaline denaturation method.The cultured alveolar macrophages（NR8383）were divided into three groups：group A（treatment with 5μg·mL-1 mtDNA）,group B（treatment with 10μg·mL-1 mtDNA）,and group C（control group）.Culture supernatant was collected after treatment for 0,3,6,12 and 24 hours,and levels of tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）and interleukin-6 （IL-6）were measured by ELISA.Results Compared with control group,mtDNA treatment significantly increased the expression of TNF-α,IL-1βand IL-6 in a dose-dependent manner after treatment for 3,6,12 and 24 hours （P〈0.01 or P〈0.05 ）.Conclusion The mtDNA can stimulate alveolar macrophages to produce TNF-α,IL-1βand IL-6 in a dose-dependent manner,suggesting that mtDNA has a proinflamma-tory effect on alveolar macrophages.