论文部分内容阅读
目的 探讨核转录因子 (NF κB)在诱导川崎病炎性细胞因子及血管内皮细胞损伤中的作用及机理。方法 建立人脐静脉内皮细胞培养模型 ,酶联免疫吸附实验 (ELISA)双抗体夹心法检测川崎病患儿外周血单个核细胞 (PBMC)活化后培养上清液白细胞介素 6 (IL 6 )、白细胞介素 1β(IL 1β)、肿瘤坏死因子α(TNF α)的分泌量 ;采用AnnexinⅤ /PI双染色法 ,流式细胞仪检测川崎病患儿PBMC培养上清液所诱导的内皮细胞凋亡率 ;凝胶电泳迁移率转换实验 (EMSA)检测所活化PBMC核因子NF κB的活性。结果 川崎病组患儿PBMC体外经刺激后IL 6、IL 1β、TNF α等炎性细胞因子的分泌均明显高于正常对照组 (P <0 0 1) ,其培养上清液所诱导的内皮细胞凋亡率 [(38 4± 7 8) %]则较正常对照组 [(2 8± 0 8) %]明显升高。分别加入足量白细胞介素 6单克隆抗体 (IL 6McAb)、肿瘤坏死因子α单克隆抗体 (TNF αMcAb)、白细胞介素 1β受体a(IL 1ra)或联合加入上述阻断剂于川崎病患儿PBMC培养上清液中 ,可不同程度地逆转川崎病患儿PBMC培养上清液所诱导内皮细胞的凋亡。川崎病患儿PBMC刺激活化后NF κB活性显著增高。NF κB抑制剂SN50 明显抑制川崎病患儿PBMC上述细胞因子的分泌 ,与此同时基本阻断川崎病患儿PBMC培养上清液所诱导内皮细
Objective To investigate the role and mechanism of nuclear factor kappa B (NF-κB) in inducing Kawasaki disease inflammatory cytokines and vascular endothelial cell injury. Methods Cultured human umbilical vein endothelial cells were cultured and the supernatants of peripheral blood mononuclear cells (PBMCs) of children with Kawasaki disease were assayed by enzyme-linked immunosorbent assay (ELISA) for detection of interleukin 6 (IL 6) The secretion of IL-1β and TNFα was detected by flow cytometry. The apoptosis of endothelial cells induced by PBMC culture supernatant was detected by AnnexinⅤ / PI double staining. The activity of NF-κB in activated PBMCs was detected by electrophoretic mobility shift assay (EMSA). Results The secretion of IL-6, IL-1β, TNFα and other inflammatory cytokines in children with Kawasaki disease group were significantly higher than those in normal control group (P <0.01) after stimulation in vitro. The endothelial cells induced by culture supernatant The rate of apoptosis was significantly higher than that of control group [(38 4 ± 7 8)%] [(28 ± 0 8)%]. (IL 6McAb), tumor necrosis factor α monoclonal antibody (TNFαMcAb), interleukin 1β receptor a (IL 1ra), or combined with the above blockers in patients with Kawasaki disease In PBMC culture supernatant, the apoptosis of endothelial cells induced by PBMC culture supernatant in children with Kawasaki disease may be reversed to varying degrees. The activation of PBMC in children with Kawasaki disease stimulated NFκB activity was significantly increased. NF-κB inhibitor SN50 significantly inhibited the secretion of cytokines in peripheral blood mononuclear cells of children with Kawasaki disease, and at the same time it basically blocked the endothelial cells induced by PBMC culture supernatant from children with Kawasaki disease