RIP2对巨噬细胞炎性活化及极性转化的调控  

Role of RIP2 in macrophage inflammatory activation and polarization transformation

作　　者：侯亮 丁彦春[2] HOU Liang;DING Yanchun(Department of Cardiology,General Hospital of the Yangtze River Shipping,Wuhan,Hubei 430000,China;Department 2 of Cardiology,Second Affiliated Hospital of Dalian Medical University,Dalian,Liaoning 116000,China)

机构地区：[1]长江航运总医院心内科,湖北省武汉市430000 [2]大连医科大学附属第二医院心内2科,辽宁省大连市116000

出　　处：《中国动脉硬化杂志》2025年第1期30-37,共8页Chinese Journal of Arteriosclerosis

基　　金：辽宁省自然科学基金项目(2019023015);武汉市卫生健康委员会项目(WX20Q10)。

摘　　要：[目的]观察受体相互作用蛋白2(RIP2)对巨噬细胞炎性活化及极性转化的影响,探讨RIP2在巨噬细胞吞噬氧化型低密度脂蛋白(ox-LDL)过程中的作用机制。[方法]用不同剂量(10、25和50 mg/L)的ox-LDL处理THP-1源性巨噬细胞24 h,及用50 mg/L ox-LDL处理THP-1源性巨噬细胞8、16及24 h,应用实时荧光定量PCR和Western blot分别检测THP-1源性巨噬细胞中RIP2 mRNA和蛋白的表达,应用ELISA检测细胞肿瘤坏死因子α(TNF-α)、单核细胞趋化蛋白1(MCP-1)的分泌。设计3对RIP2 siRNA,应用hiperfict转染试剂转染RIP2 siRNA进入细胞,应用实时荧光定量PCR和Western blot检测THP-1源性巨噬细胞转染后RIP2 mRNA和蛋白的表达,从而筛选最适siRNA转染浓度和最有效的一对siRNA。用最有效RIP2 siRNA转染细胞后,50 mg/L ox-LDL处理24 h,应用ELISA检测TNF-α、MCP-1、白细胞介素10(IL-10)、白细胞介素12(IL-12)的分泌,应用实时荧光定量PCR检测诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg-1)的表达,应用流式细胞术检测细胞表面抗原CD86、CD80和CD163的表达。[结果]ox-LDL以剂量和时间依赖的方式诱导巨噬细胞中RIP2的表达,随着ox-LDL处理剂量及时间的增加,RIP2 mRNA及蛋白的表达增加,其中50 mg/L组RIP2蛋白的表达是对照组的7.6倍,24 h组RIP2蛋白的表达是对照组的17.9倍(P<0.001)。ELISA结果显示,随着ox-LDL处理剂量及时间的增加,TNF-α和MCP-1的分泌升高(P<0.05)。RIP2 siRNA转染细胞后,ELISA检测结果显示,ox-LDL组TNF-α、MCP-1、IL-10的分泌是对照组的2.4倍、2.9倍、1.8倍(P<0.01),IL-12的分泌较对照组下降了34.2%(P<0.01);siRNA+ox-LDL组TNF-α、MCP-1、IL-10的分泌较ox-LDL组分别下降了37.4%、45.3%、27.4%(P<0.01),IL-12的分泌增加了31.4%(P<0.05)。流式细胞术和实时荧光定量PCR结果显示,ox-LDL组CD86、CD80和iNOS mRNA的表达分别是对照组的14.2倍、33.8倍和4.5倍,CD163和Arg-1 mRNA的表达较对照组分别下降了33.4%和43.0%(P<0.05)Aim To observe the effects of receptor interacting protein 2(RIP2)on macrophage inflammatory activation and polarity transformation,and to explore the mechanism of RIP2 in macrophage phagocytosis of oxidized low density lipoprotein(ox-LDL).Methods THP-1 derived macrophages were treated with different doses(10,25 and 50 mg/L)of ox-LDL for 24 hours,and treated with 50 mg/L ox-LDL for 8,16 and 24 hours.Real-time quantitative PCR and Western blot were used to detect the expression of RIP2 mRNA and protein in THP-1 derived macrophages,and ELISA was used to detect the secretion of tumor necrosis factor-α(TNF-α)and monocyte chemoattractant protein-1(MCP-1).Three pairs of RIP2 siRNA were designed,transfecting them into cells using hiperfict transfection reagent,real-time quantitative PCR and Western blot were used to detect the expression of RIP2 mRNA and protein in THP-1 derived macrophages after transfection,in order to screen for the optimal siRNA transfection concentration and the most effective pair of siRNA.After transfection with the most effective RIP2 siRNA,cells were treated with 50 mg/L ox-LDL for 24 hours,ELISA was used to detect the secretion of TNF-α,MCP-1,interleukin-10(IL-10)and interleukin-12(IL-12),real-time quantitative PCR was used to detect the expression of inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1),flow cytometry was used to detect the expression of cell surface antigens CD86,CD80 and CD163.Results Ox-LDL induced the expression of RIP2 in macrophages in a dose-dependent and time-dependent manner.With the increase of ox-LDL treatment dose and time,the expression of RIP2 mRNA and protein increased.Among them,the expression of RIP2 protein in the 50 mg/L group was 7.6 times of the control group,and the expression of RIP2 protein in the 24 h group was 17.9 times of the control group(P<0.001).The ELISA results showed that with the increase of ox-LDL treatment dose and time,the secretion of TNF-αand MCP-1 increased(P<0.05).After transfection of RIP2 siRNA into cells,ELISA results show

关 键 词：受体相互作用蛋白2 氧化型低密度脂蛋白 动脉粥样硬化 巨噬细胞 

分 类 号：R5[医药卫生—内科学] R363[医药卫生—临床医学]