过表达锌指蛋白A20可抑制肺泡巨噬细胞炎症反应  

A20 regulates the inflammatory responses of alveolar macrophage

作　　者：朱晓丹[1] 王颖[1] 毕晶[1] 童琳[1] 刘洁[1] 宋元林[1] 白春学[1] ZHU Xiao-dan WANG Ying BI Jing TONG Lin LIU Jie SONG Yuan-lin BAI Clum-xue(D epartm ent of R esp iratory M edicine, Zhongshan H o sp ital, Fudan U n iv ersity , Shanghai 2 0 0 0 3 2 , Chin)

机构地区：[1]复旦大学附属中山医院呼吸内科,上海200032

出　　处：《中国临床医学》2016年第6期715-719,共5页Chinese Journal of Clinical Medicine

基　　金：国家自然科学基金青年基金(81300055;81500025);国家自然科学基金重点项目(30930090)~~

摘　　要：目的:通过建立A20过表达肺泡巨噬细胞株,研究A20对肺泡巨噬细胞炎症反应的影响及调控机制。方法:构建携带A20基因的慢病毒载体,转染大鼠肺泡巨噬细胞(NR8383),筛选出稳定过表达A20基因的细胞株并行体外培养。向培养基中加入脂多糖(LPS,1μg/mL)进行干预,并于刺激后0.5、1、2、4h收集上清液及细胞。ELISA法测定上清液中细胞因子(TNF-α、IL-1β)及核因子κB(NF-κB)活性。Western印迹方法检测A20蛋白及核内p65含量。实时荧光定量PCR法测定A20mRNA含量。结果:LPS刺激后,A20过表达组(A20组)及正常对照组(VEC组)A20蛋白及mRNA含量都升高,并于1h达高峰,之后逐渐下降;且A20组较VEC组A20水平明显升高(P<0.05)。与VEC组相比,A20组培养上清液中细胞因子(TNF-α、IL-1β)水平明显降低(P<0.05);NF-κB DNA结合活性及核内p65含量也降低(P<0.05)。结论:A20能够抑制肺泡巨噬细胞NF-κB活性及TNF-α、IL-1β分泌,进而抑制肺泡巨噬细胞炎症反应活性。Objective ： T o study th e effects of A 20 on the inflam m atory response of alveolar m acrophages and itsregulation m echanism through establish ing A 20 ov er-exp ressing alveolar m acrophage cell lines. Methods ： Lentivirus-m ediatedexp ression v ector carrying A 20 gene was con stru cted , then it was transfected into ra t alveolar m acrophage cell lines （ N R 8 3 8 3 ） ,and th e cell lines w hich stably overexpressed A 20 gene w ere screened and cultured in v itr o . L ipopolysaccharide （ L P S , 1 y.g /m L ） w as added into the medium to interven e, th e culture supernatants and cells w ere collected 0. 5 ,1 ,2 , 4 hours afterstim u lation , E L IS A m ethod w as used to determ ine the activity of cytokines （T N F -a IL-1β） and nuclear fa cto r （N F-kB ）.W estern blotting m ethod w as used to d etect A 20 protein and nuclear p65 co n ten t, and real-tim e flu orescence quantitative P C Rm ethod was used to determ ine the content of A 20 m R N A . Results： A fte r L P S stim u lation , the levels of A 20 protein andm R N A in A 20 ov erexpression group （A 2 0 group） and th e norm al control group （ V E C group） b o th increased and reached thepeak at 1 h , and then gradually decreased. T h e A 20 level of A 20 group was significantly higher than that of V E C group （P〈C0. 0 5 ）. Com pared w ith V E C group , th e levels of cytokines （T N F -a IL-1β） in culture supernatants of A 20 group significantlyreduced （P 〈 . 0 5 ） , and the D N A binding activity of NF-kB and nuclear p65 content of A 20 group also significantly reduced（P〈 0. 0 5 ）. Conclusions： A 20 can inhibit the activity of NF-kB and the secretion of T N F -a and IL -1β, and then inhibit theinflam m atory reaction activity of alveolar m acrop hages, thereby reducing the inflam m atory response of acu te resp iratoryd istress syndrom e （A R D S ）.

关 键 词：急性呼吸窘迫综合征 A20蛋白 肺泡 巨噬细胞 炎症反应 

分 类 号：R563.8[医药卫生—呼吸系统]