山东医药
山東醫藥
산동의약
SHANDONG MEDICAL JOURNAL
2015年
6期
11-13
,共3页
谈诚%韩勍%朱纯亮%李喆倩%刘丽莎
談誠%韓勍%硃純亮%李喆倩%劉麗莎
담성%한경%주순량%리철천%류려사
川崎病%核因子-κB%基质金属蛋白酶9%外周血单个核细胞
川崎病%覈因子-κB%基質金屬蛋白酶9%外週血單箇覈細胞
천기병%핵인자-κB%기질금속단백매9%외주혈단개핵세포
Kawasaki disease%nuclear factor-kappaB%matrix metalloproteases 9%peripheral blood mononuclear cells
目的：探讨核细胞核因子-κB（NF-κB）和基质金属蛋白酶9（MMP-9）在川崎病发生、发展中的作用及其机制。方法研究对象为川崎病患儿21例（川崎病组）、呼吸道感染患者18例（阳性对照组）、正常查体者17例（正常对照组）。采用密度梯度离心法无菌分离各组外周血PBMCs自然培养，采用RT-PCR法和Westen blot法测定细胞中NF-κB p65 mRNA和蛋白，采用ELISA方法测定细胞上清液中MMP-9蛋白。将川崎病组外周血PBMCs体外培养并分为川崎病1、2、3组，川崎病1组继续自然培养，川崎病2组加入终浓度为20 nmol／L的NF-κB激动剂PMA，川崎病3组同时加入PMA（浓度同上）及终浓度为100μmol／L的NF-κB抑制剂 PDTC，孵育2 h后收集上清液和细胞，分别采用RT-PCR法和Westen blot法测定NF-κB p65 mRNA和蛋白表达，采用ELISA法测定细胞上清液中MMP-9蛋白水平。结果川崎病组PBMCs中NF-κB p65 mRNA、蛋白及上清液MMP-9表达均高于阳性对照组和健康对照组（ P均＜0．05）。川崎病1、3组PBMCs中NF-κB p65蛋白及上清液中MMP-9表达水平均低于川崎病2组（P均＜0．05）。结论急性期川崎病患儿外周血PBMCs 中NF-κB和细胞上清液中MMP-9表达增强；二者可能参与了川崎病的发生发展过程。。
目的：探討覈細胞覈因子-κB（NF-κB）和基質金屬蛋白酶9（MMP-9）在川崎病髮生、髮展中的作用及其機製。方法研究對象為川崎病患兒21例（川崎病組）、呼吸道感染患者18例（暘性對照組）、正常查體者17例（正常對照組）。採用密度梯度離心法無菌分離各組外週血PBMCs自然培養，採用RT-PCR法和Westen blot法測定細胞中NF-κB p65 mRNA和蛋白，採用ELISA方法測定細胞上清液中MMP-9蛋白。將川崎病組外週血PBMCs體外培養併分為川崎病1、2、3組，川崎病1組繼續自然培養，川崎病2組加入終濃度為20 nmol／L的NF-κB激動劑PMA，川崎病3組同時加入PMA（濃度同上）及終濃度為100μmol／L的NF-κB抑製劑 PDTC，孵育2 h後收集上清液和細胞，分彆採用RT-PCR法和Westen blot法測定NF-κB p65 mRNA和蛋白錶達，採用ELISA法測定細胞上清液中MMP-9蛋白水平。結果川崎病組PBMCs中NF-κB p65 mRNA、蛋白及上清液MMP-9錶達均高于暘性對照組和健康對照組（ P均＜0．05）。川崎病1、3組PBMCs中NF-κB p65蛋白及上清液中MMP-9錶達水平均低于川崎病2組（P均＜0．05）。結論急性期川崎病患兒外週血PBMCs 中NF-κB和細胞上清液中MMP-9錶達增彊；二者可能參與瞭川崎病的髮生髮展過程。。
목적：탐토핵세포핵인자-κB（NF-κB）화기질금속단백매9（MMP-9）재천기병발생、발전중적작용급기궤제。방법연구대상위천기병환인21례（천기병조）、호흡도감염환자18례（양성대조조）、정상사체자17례（정상대조조）。채용밀도제도리심법무균분리각조외주혈PBMCs자연배양，채용RT-PCR법화Westen blot법측정세포중NF-κB p65 mRNA화단백，채용ELISA방법측정세포상청액중MMP-9단백。장천기병조외주혈PBMCs체외배양병분위천기병1、2、3조，천기병1조계속자연배양，천기병2조가입종농도위20 nmol／L적NF-κB격동제PMA，천기병3조동시가입PMA（농도동상）급종농도위100μmol／L적NF-κB억제제 PDTC，부육2 h후수집상청액화세포，분별채용RT-PCR법화Westen blot법측정NF-κB p65 mRNA화단백표체，채용ELISA법측정세포상청액중MMP-9단백수평。결과천기병조PBMCs중NF-κB p65 mRNA、단백급상청액MMP-9표체균고우양성대조조화건강대조조（ P균＜0．05）。천기병1、3조PBMCs중NF-κB p65단백급상청액중MMP-9표체수평균저우천기병2조（P균＜0．05）。결론급성기천기병환인외주혈PBMCs 중NF-κB화세포상청액중MMP-9표체증강；이자가능삼여료천기병적발생발전과정。。
Objective To explore the activation of nuclear factor-kappaB ( NF-κB ) and matrix metalloproteases 9 (MMP-9) in peripheral blood mononuclear cells (PBMCs)in children with Kawasaki disease and the relationship between NF-κB and MMP-9.Methods PBMCs were isolated and purified from blood of fifty-six children including 21children with KD, 18 children with general inflammatory and 17 normal ones by density gradient centrifugation ,and were cultured for one hour after cell concentration adjusted to 106/mL.Then both KD group and Control group were divided into three groups ,the first group was cultured naturally , the second one was stimulated by phorbol 12-myristate 13-acetate(PMA), and the third one was stimulated by PMA and pyrrolidine dithiocarbamate ( PDTC) .PBMCs were cultured for 2 h.The mRNA and pro-tein levels of NF-κB p65 in PBMCs in each groups were measured by RT-PCR and Western blot , and the protein levels of MMP-9 were measured by ELISA .Results The mRNA and protein levels of NF-κB p65 and MMP-9 in KD group were much higher than that in other groups;Stimulating PBMCs by PMA ,the mRNA and protein levels of NF-κB p65 and MMP-9 were markedly increased in each group , and there was a significant difference in each group (P<0.05); Using PDTC can reduce the activation of NF-κB in PBMCs, and the protein levels of MMP-9 were significantly reduced by using PDTC in KD group.Conclusions The activity of NF-κB and MMP-9 in PBMCs in patients with acute KD is distinctly increased;The specific inhibitor of NF-κB can inhibit it's activation and the expression of MMP-9 obviously.It suggested that the NF-κB pathway can lead to the activation of MMP-9 in acute KD, and proper application of specific inhibitor of NF-κB may be a new choice for treating KD .
