摘要
目的:研究白细胞介素1 Ⅱ型受体(IL-1RⅡ)和白细胞介素1受体辅助蛋白(IL-1RAcP)重组质粒对大鼠实验性自身免疫性心肌炎(EAM)的作用及其机制。方法:构建体内导入用重组质粒pCAGGS-IL-1RⅡ和pCAGGSIL-1RAcP,并以pCAGGS-SP(signal peptide)作为对照质粒。将雄性Lewis大鼠分成4组:对照(control)组(未免疫未注射质粒的大鼠,n=5)、EAM+SP组(免疫的大鼠尾静脉注射对照质粒,n=9)、EAM+IL-1RⅡ组(免疫的大鼠尾静脉注射IL-1RⅡ质粒,n=8)和EAM+IL-1RⅡ+IL-1RAcP组(免疫的大鼠尾静脉注射IL-1RⅡ和IL-1RAcP质粒,n=7)。第0天免疫大鼠造模;第6天应用流体动力学方法尾静脉注射重组质粒;第17天处死大鼠进行疗效评估,评估指标包括组织病理学、心脏彩超检测、心脏重量/体重比值、心肌中心衰标志物心房钠尿肽(ANP)和脑钠尿肽(BNP)及炎症因子的表达。构建体外细胞转染用重组质粒pUC19-IL-1RⅡ-actin和pUC19-IL-1RAcP-tub,转染入Cos7细胞获得培养上清液,将含有IL-1RⅡ和IL-1RAcP的培养上清液加至脂多糖(LPS)诱导的H9c2细胞中,RT-qPCR检测炎症因子的表达变化。将构建的重组质粒pEGFP-IL-1RⅡ-actin和pEGFP-IL-1RAcP-tub转染入Cos7细胞,免疫共沉淀(Co-IP)检测目的蛋白的形成。结果:与EAM+SP组相比,EAM+IL-1RⅡ组和EAM+IL-1RⅡ+IL-1RAcP组大鼠心肌炎症程度均有显著减轻,左心室收缩期内径显著缩小(P<0.05),心肌中ANP、BNP、TNF-α、IL-2、IFN-γ和TGF-β的表达水平显著降低(P<0.01),IL-4的表达水平显著升高(P<0.01)。在H9c2细胞中,与LPS组比较,LPS+IL-1RⅡ组和LPS+IL-1RⅡ+IL-1RAcP组TGF-β和IL-6的表达水平显著降低(P<0.01),IL-10的表达水平显著升高(P<0.05);与LPS+IL-1RⅡ组比较,LPS+IL-1RⅡ+IL-1RAcP组TNF-α和IL-2的表达水平显著降低(P<0.05),IL-13的水平显著升高(P<0.01)。Co-IP检测到IL-1RⅡ与IL-1RAcP相互结合并形成了一个新的蛋白,为异源二聚体。结论:IL-1RⅡ和IL-1RAcP重组质粒导入可有效缓�
AIM:To evaluate the effects of recombinant plasmids encoding interleukin-1 type Ⅱ receptor(IL-1RⅡ)and interleukin-1 receptor accessory protein(IL-1 RAcP)on rat experimental autoimmune myocarditis(EAM)and the possible mechanism.METHODS:The recombinant plasmids pCAGGS-IL-1RⅡ and pCAGGS-IL-1 RAcP were constructed,and pCAGGS-SP(signal peptide)served as the control plasmid.Male Lewis rats(n=29)were divided into 4 groups:control group(rats without immunization or injection,n=5),EAM+SP group(immunized rats injected with pCAGGS-SP,n=9),EAM+IL-1RⅡ group(immunized rats injected with pCAGGS-IL-1RⅡ,n=8)and EAM+IL-1RⅡ+IL-1RAcP group(immunized rats injected with pCAGGS-IL-1RⅡ and pCAGGS-IL-1 RAcP,n=7).The rats were immunized to induce EAM on day 0,and injected with recombinant plasmids by hydrodynamics-based delivery on day 6.Echocardiography was performed,and the rats were killed on day 17.The ratio of heart weight to body weight(HW/BW)was evaluated,and the histopathological changes of the myocardial tissues were observed by HE staining.The mRNA expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and inflammatory factors in the myocardial tissues was detected by RT-qPCR.Recombinant plasmids pUC19-IL-1RⅡ-actin and pUC19-IL-1 RAcP-tub were transfected into Cos7 cells,and the culture supernatants were collected and added to lipopolysaccharide(LPS)-induced H9c2 cells.The expression of inflammatory genes were detected by RT-qPCR.Recombinant plasmids pEGFP-IL-1RⅡ-actin and pEGFP-IL-1 RAcP-tub were transfected into the Cos7 cells to identify the formation of IL-1RⅡ/IL-1 RAcP heterodimer by co-immunoprecipitation(Co-IP).RESULTS:Compared with EAM+SP group,injection with plasmids effectively attenuated EAM in EAM+IL-1RⅡ group and EAM+IL-1RⅡ+IL-1 RAcP group,as indicated by the decreases in HW/BW,left ventricular end-systolic diameter,and myocardial expression of ANP,BNP,TNF-α,IL-2,IFN-γ and TGF-β,and the increase in expression of IL-4 in the hearts.In LPS-induced H9c2 cells,compared wit
作者
常贺
宋颖
刘春晓
CHANG He;SONG Ying;LIU Chun-xiao(Department of Geriatrics,Xiang'an Hospital of Xiamen University,Xiamen 361100,China;Xiamen Cardiovascular Hos-pital,Xiamen University,Xiamen 361014,China;Xiamen Branch of Zhongshan Hospital,Fudan University,Xiamen 361015,China.)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2020年第10期1729-1738,共10页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81270294)
厦门大学附属翔安医院引进高层次人才科研启动基金资助项目(No.PM201809170018)。
