siRNA抑制高迁移率族蛋白B1对机械牵张诱导肺泡巨噬细胞细胞因子表达谱改变的影响  

Effect of inhibition of HMGB1 expression by small interfering RNA on mechanical stretch-induced cytokines release in alveolar macrophages

作　　者：罗兵[1] 丁宁[1] 许立新[1] 佘守章[1] 

机构地区：[1]广州市第一人民医院麻醉科,广东广州510180

出　　处：《中国急救医学》2013年第9期837-840,共4页Chinese Journal of Critical Care Medicine

基　　金：国家自然科学基金面上项目（81272136）;广东省科技计划项目（2010B031600011）;广州市科技计划重点项目（2012J4100034）;广州市医药卫生科技重点项目（20121A021001）

摘　　要：目的 探讨小干扰RNA（small interfering RNA,siRNA）抑制高迁移率族蛋白B1（high mobility group box 1 protein,HMGB1）基因的表达对机械牵张诱导肺泡巨噬细胞（alveolar macrophage,AM）细胞因子释放的影响.方法 将HMGB1 siRNA真核表达质粒（siHMGB1）在脂质体介导下转染AM,以转染siRNA-A质粒的细胞作为阴性对照.实验分为4组,siHMGB1未牵张组、siHMGB1牵张组、siRNA-A未牵张组和siRNA-A牵张组.将牵张组Bioflex弹性膜细胞培养板置于Flexercell 4000T应力加载系统中,给予细胞施加20%牵张应变,牵张频率为30次/min,牵拉/松弛比为1：1,牵张时间为24 h.采用RT-PCR和Western blot检测HMGB1 mRNA和蛋白表达,应用LiquiChip液相蛋白芯片系统检测细胞培养上清中细胞因子浓度.结果 转染后与siRNA-A转染组比较,siHMGB1转染组细胞HMGB1 mRNA和蛋白表达均显著下降（P〈0.05）;siHMGB1转染细胞可显著抑制机械牵张诱导的IL-1 、IL-6、MIP-2、MCP-1、IFN-γ和IP-10释放（P〈0.05）.结论 应用RNA干扰（RNAi）技术可以有效抑制HMGB1 mRNA和蛋白的表达,进而抑制机械牵张诱导的细胞因子释放,为机械通气所致肺损伤（ventilator-induced lung injury,VILI）的基因治疗提供新思路.Objective To explore the effects of inhibition of HMGB1 expression by small interfering RNA （siRNA） on mechanical stretch - induced cytokines release in alveolar macrophages （AMs）. Methods The eukaryotic expression vector HMGB1 siRNA was transfected into AMs by lipofectamine2000. Cells transfected with siRNA - A were used as negative control. AMs were divided into siHMGB1 unstretched group, siHMGB1 stretched group, siRNA - A unstretched group and siRNA -A stretched group. Stretched AMs were subjected to 20% elongation by Flexercell 4000T cell stress system for 24 h. HMGB1 mRNA and protein expression was detected by RT - PCR and Western blot. Cell culture supernatant was collected to detect the levels of IL - 1, IL - 6, MIP - 2, MCP - 1, IFN - γ and IP - 10 by using LiquiChip system. Results The recombinant plasmid HMGB1 siRNA was successfully transfected into AMs and the introduction of HMGB1 siRNA efficiently inhibited the expression of the HMGB1 mRNA and protein according to the results of RT - PCR and Western blot, with a significant difference between the HMGB1 siRNA transfected group and control group （P 〈 0.05）. The levels of IL-1, IL-6, MIP-2, MCP-1, IFN-T and IP-10 secreted by AMs subjected to mechanical stretch were significantly lower in HMGB1 siRNA transfected group than in control group （ P 〈 0.05 ）. Conclusion siRNA targeting HMGB1 mRNA can specifically suppress HMGB1 gene expression and effectively inhibit mechanical stretch - induced cytokines release in AMs. This study provides preliminary results in the searching of RNAi therapy for ventilator - induced lung injury （VILI）.

关 键 词：机械牵张 肺泡巨噬细胞 细胞因子 呼吸机所致肺损伤 RNA干扰 

分 类 号：R631.02[医药卫生—外科学]