DDIAS在川崎病患儿血液中的表达及意义

The Expression and Significance of DDIAS in the Blood of Children with Kawasaki Disease

目的:研究DNA损伤诱导凋亡抑制因子(DDIAS)在川崎病(KD)患儿血液中的表达及意义。方法:通过彩色多普勒超声心动图检查96例KD患儿的冠状动脉损伤(CAL)情况,根据CAL存在情况将患儿分为CAL组和非CAL组。选取我院同期体检的30例健康儿童作为对照组。通过ELISA试剂盒检测受试者外周血中DDIAS的水平。将靶向DDIAS的小干扰RNA(siRNA)(DDIAS-siRNA)和阴性对照siRNA(NC-siRNA)转染到人冠状动脉内皮细胞(HCAEC)中,并用10 ng/mL TNF-α处理细胞。通过RT-PCR分析细胞中的TNF-α、IL-6和HO-1 mRNA水平,通过Western blot分析DDIAS、NF-κB p65和I-κBα的蛋白水平。此外,评估了各组HCAEC细胞中的超氧化物和谷胱甘肽(GSH)含量及其与单核细胞的粘附。结果:与对照组比较,KD组的DDIAS水平显著升高(P<0.001)。与非CAL组比较,CAL组的DDIAS水平显著升高(P<0.01)。外周血DDIAS诊断KD的AUC、敏感性和特异性依次为0.747、65.62%和80.65%,外周血DDIAS诊断CAL的AUC、敏感性和特异性依次为0.733、63.83%和75.51%。与NC-siRNA+TNF-α组相比,DDIAS-siRNA+TNF-α组HCAEC细胞中的TNF-αmRNA表达水平降低了51.45%,IL-6 mRNA表达水平降低了59.46%,细胞核NF-κB p65的蛋白表达水平降低了26.40%,细胞质I-κBα的蛋白水平升高了91.30%(P<0.05)。与NC-siRNA+TNF-α组相比,DDIAS-siRNA+TNF-α组粘附实验的相对荧光强度降低了53.42%(P<0.05)。与NC-siRNA+TNF-α组相比,DDIAS-siRNA+TNF-α组HCAEC细胞中的超氧化物相对荧光强度降低了35.38%,HO-1 mRNA水平升高了1.35倍,GSH水平升高了94.59%(P<0.05)。结论:DDIAS对KD及CAL均有较高的诊断价值,下调DDIAS减轻TNF-α诱导的HCAEC细胞损伤。

Objective:To study the expression and significance of DNA damage-induced apoptosis inhibitor(DDIAS)in the blood of children with Kawasaki disease(KD).Methods:The coronary artery injury(CAL)of 96 children with KD was examined by color Doppler echocardiography,and the children were divided into CAL group and non-CAL group according to the presence of CAL.30 healthy children who received physical examination in our hospital during the same period were selected as the control group.The level of DDIAS in the peripheral blood of subjects was detected by an ELISA kit.Small interfering RNA(siRNA)targeting DDIAS(DDIAS-siRNA)and negative control siRNA(NC-siRNA)were transfected into human coronary artery endothelial cells(HCAEC),and the cells were treated with 10 ng/mL TNF-α.The levels of TNF-α,IL-6 and HO-1 mRNA in the cells were analyzed by RT-PCR,and the protein levels of DDIAS,NF-κB p65 and I-κBαwere analyzed by Western blot.In addition,the superoxide and glutathione(GSH)content in HCAEC cells of each group and their adhesion to monocytes were evaluated.Results:Compared with the control group,the level of DDIAS in the KD group was significantly increased(P<0.001).Compared with the non-CAL group,the level of DDIAS in the CAL group was significantly increased(P<0.01).The AUC,sensitivity and specificity of peripheral blood DDIAS for KD diagnosis were 0.747,65.62%and 80.65%,respectively.The AUC,sensitivity and specificity of peripheral blood DDIAS for diagnosing CAL were 0.733,63.83%and 75.51%,respectively.Compared with the NC-siRNA+TNF-αgroup,the TNF-αmRNA level in the HCAEC cells of the DDIAS-siRNA+TNF-αgroup was reduced by 51.45%,the IL-6 mRNA level was reduced by 59.46%,the protein level of nuclear NF-κB p65 was reduced by 26.40%,and the protein level of cytoplasmic I-κBαincreased by 91.30%(P<0.05).Compared with the NC-siRNA+TNF-αgroup,the relative fluorescence intensity of the adhesion experiment in the DDIAS-siRNA+TNF-αgroup was reduced by 53.42%(P<0.05).Compared with the NC-siRNA+TNF-αgroup,the relative fluorescence intensity of superoxide in HCAEC cells in the DDIAS-siRNA+TNF-αgroup was reduced by 35.38%,the level of HO-1 mRNA was increased by 1.35 times,and the level of GSH was increased.94.59%(P<0.05).Conclusion:DDIAS has high diagnostic value for KD and CAL.Down-regulation of DDIAS reduces the damage of HCAEC cells induced by TNF-α.