CAJ | 학술논문

目的观察阻断IgG-FcγR结合的短肽tg19320对粒细胞与内皮细胞黏附以及内皮细胞细胞问黏附分子1(ICAM-1)表达的影响,并探讨其作用机制.方法培养人脐静脉内皮细胞(HUVEC).提纯活动期血管炎患者血清抗中性粒细胞胞质抗体(ANCA)IgG.以多肽固相合成tg19320.分离健康人新鲜外周血中性粒细胞.分别以肿瘤坏死因子α(TNF-α)、健康人IgG、ANCA IgG及ANCA IgG+tg19320处理HUVEC,用细胞直接计数法检测粒细胞与内皮细胞间的黏附率;应用Western印迹及实时定量PCR方法检测HUVEC ICAM-1蛋白和mRNA表达;ELISA检测细胞培养上清液中可溶性ICAM-1(sICAM-1)的水平;Western印迹检测HUVEC磷酸化核因子κB抑制物(p-IκB)的表达.结果 ANCA IgG显著上调中性粒细胞与内皮细胞问的黏附率(P＜0.05),但ANCA IgG+tg19320组较ANCA IgG组黏附率显著降低(P＜0.05).ANCA IgG组与健康人IgG组相比,HUVEC ICAM-1 mRNA及蛋白表达显著增加(P＜0.05).tg19320分别从mRNA和蛋白水平阻断ANCA对ICAM-1的作用(P＜0.05),并显著降低ANCA IgG引起的细胞培养上清液中sICAM水平增高(P＜0.05).ANCA IgG增加HUVEC p-IκB表达,tg19320显著降低p-IκB的表达.结论 tg19320通过抑制IκB磷酸化进而干预NF-κB活化;抑制ANCA IgG对内皮细胞与中性粒细胞问黏附的促进作用,并阻断ICAM-1上调表达.短肽tg19320对原发性小血管炎具有体外保护作用.
목적 관찰조단IgG-FcγR결합적단태tg19320대립세포여내피세포점부이급내피세포세포문점부분자1(ICAM-1)표체적영향,병탐토기작용궤제.방법 배양인제정맥내피세포(HUVEC).제순활동기혈관염환자혈청항중성립세포포질항체(ANCA)IgG.이다태고상합성tg19320.분리건강인신선외주혈중성립세포.분별이종류배사인자α(TNF-α)、건강인IgG、ANCA IgG급ANCA IgG+tg19320처리HUVEC,용세포직접계수법검측립세포여내피세포간적점부솔;응용Western인적급실시정량PCR방법검측HUVEC ICAM-1단백화mRNA표체;ELISA검측세포배양상청액중가용성ICAM-1(sICAM-1)적수평;Western인적검측HUVEC린산화핵인자κB억제물(p-IκB)적표체.결과 ANCA IgG현저상조중성립세포여내피세포문적점부솔(P＜0.05),단ANCA IgG+tg19320조교ANCA IgG조점부솔현저강저(P＜0.05).ANCA IgG조여건강인IgG조상비,HUVEC ICAM-1 mRNA급단백표체현저증가(P＜0.05).tg19320분별종mRNA화단백수평조단ANCA대ICAM-1적작용(P＜0.05),병현저강저ANCA IgG인기적세포배양상청액중sICAM수평증고(P＜0.05).ANCA IgG증가HUVEC p-IκB표체,tg19320현저강저p-IκB적표체.결론 tg19320통과억제IκB린산화진이간예NF-κB활화;억제ANCA IgG대내피세포여중성립세포문점부적촉진작용,병조단ICAM-1상조표체.단태tg19320대원발성소혈관염구유체외보호작용.
Objective To investigate the effects of tg19320,a small peptide,interfering with IgG-FcγR interaction on the adhesion of neutrophils to endothelium and the expression of intercellular adhesion molecule 1 (ICAM-1)in endothelial cells and its possible mechanism.Methods Tg19320 was prepared by solid-phase peptide synthesis.ANCA IgG was isolated from the serum of active ANCA-associated systemic vasculitis(AASV)patients.When primary human umbilical vein endothelial cells(HUVEC)grew into connuence in cytokine-free eonditions,the cells were stimulated with TNF-α,human normal IgG,ANCA IgG and ANCA IgG+tg19320 respectively.HUVEC were pretreated with tg19320 for 45 minutes before being stimulated by ANCA IgG.Non-activated neutrophils was added to treat HUVEC and adhesion was measured by cell count.The expression of ICAM-1 mRNA and protein was assessed by real-time PCR and Western blot respectively.Soluble ICAM-1(sICAM-1)was determined using ELISA technique.Phosphorylation of IκB-α was assessed by Western blot. Results ANCA IgG significantly up-regulated the expression of ICAM-1 in HUVEC and promoted sICAM-1 release(P＜0.05),and TNF-α enhanced the effect of ANCA.These effects were almost completely abolished by tgl9320 both at protein and mRNA level.Furthermore,ANCA IgG increased the IκB-α phosporylation in HUVEC and tg19320could inhibit the effect. Conclusions ANCA IgG can modulate the expression of ICAM-1 and sICAM-1 release in endothelial cells.FcγR probably play a critical role in the ICAM-1 expression up-regulated by ANCA,which is mediated in part through NF-κB signaling pathway.Tg19320 has protective effect on endothelium in AASV in vitro.