川崎病急性期中性粒细胞功能及S100蛋白表达的变化

目的 观察中性粒细胞在川崎病（KD）急性期的功能及S100 A8/A9蛋白表达的变化，并探讨其意义。方法依据纳入标准和排除标准选取2006年11月至2007年7月在复旦大学附属儿科医院住院的KD患儿为研究对象，选取同期手术患儿为本研究对照组。通过二氢若丹明荧光染色法分析KD患儿中性粒细胞功能，并用荧光定量PCR法检测KD患儿中性粒细胞S100 A8/A9 mRNA表达。结果共纳入KD患儿32例,其中男19例,女13例,年龄2个月至7岁2个月，平均（2.1±1.9）岁。冠状动脉损害者6例，无冠状动脉损害者26例；对照组手术患儿20例。KD急性期中性粒细胞明显活化，使用IVIG治疗后中性粒细胞活化百分比下降。KD急性期中性粒细胞S100 A8/A9 mRNA的表达增加；IVIG治疗后，无冠状动脉损害患儿，S100 A8/A9 mRNA表达明显降低，冠状动脉损害患儿S100 A8/A9 mRNA表达升高。结论 KD急性期中性粒细胞活化，并存在相关的活化蛋白高表达，提示中性粒细胞可能参与了KD的发病机制。且在冠状动脉损害患儿中性粒细胞S100 A8/A9 mRNA持续高表达，提示中性粒细胞可能参与冠状动脉损害。

Changes of the expression of S100 protein and the function of neutrophils in acute Kawasaki disease

Objective Kawasaki disease (KD) is one of the most common acute vasculitis syndromes in children, and mostly affects arteries, particularly, coronary arteries. Currently, its etiology and etiopathology are still largely unknown. S100 A8, S100 A9 are members of calcium binding protein family, and mostly secreted by activated neutrophils and monocytes. Several studies have demonstrated that they play vital roles in acute inflammation. However, the expression of these two proteins in KD is rarely studied. This project was to investigate functional change of neutrophils in KD and its relevance to the progress of the disease.Methods According to the criteria set in Japan 2002, 32 patients with KD were enrolled in the study. Meanwhile, 20 surgery patients with similar age were enrolled as negative controls. Blood samples were collected at the time of diagnosis before the initiation of intravenous immune globulin(IVIG) treatment, and 1 week after IVIG treatment. To evaluate the activation of neutrophils, partial patients underwent DHR assay. 0.2 mL of heparin anticoagulated venous blood was collected, 50μL of which was incubated in phosphate buffer solution(PBS), phorbol myristate acetate(PMA) for 15 minutes, N Formyl Met Leu Phe(fMLP) for 5 minutes at 37℃ respectively, then incubated with 25 μL of dihydrorhodamine(DHR) for 5 minutes at 37℃. After that erythrocytes were removed by adding 2 mL of hemolysis and cells were washed once with PBS, re suspended in 300-500 μL of PBS and then assayed by flow cytometry. Total RNA was extracted from neutrophils and preserved by Trizol reagent. The purity and quantity of RNA were determined according to ultraviolet absorbance at 260nm and 280nm.Gene expression of S100A8 and S100A9 in neutrophils was analyzed by real time fluorescence quantitative polymerase chain reaction.ResultsAfter incubation with PBS and fMLP, the proportions of activated neutrophils in 11 cases before IVIG treatment were higher than those of the control (PBS: P=0.033; fMLP: P=0.000), decreased after IVIG treatment in 6 self control cases (PBS: from 33.5±21.4 to 16.0±11.7, P=0.109; fMLP: from 78.4±15.9 to 30.1±21.1, P=0.001), reached the control level (PBS:P=0.149; fMLP:P=0.867). Before IVIG treatment in KD patients, the expression of S100 A8,S100 A9 in neutrophils was significantly higher than that of the control (P=0.000). In patients without conronary artery lesions(CALs), the expression levels of S100 A8,S100 A9 mRNA in neutrophils were 6.92±0.59 and 6.80±0.51 respectively. After IVIG treatment, the expressions of these two genes decreased to 6.16±0.81 and 6.00±0.77 respectively (P=0.001 and 0.000). However, in patients with CALs, the expression levels of S100 A8,S100 A9 mRNA were 5.97±0.32 and 6.14±0.45 respectively. After IVIG treatment, the expressions of these two genes increased to 6.66±0.63 and 6.58±0.45 respectively(P=0.038 and 0.12). Conclusions In acute Kawasaki disease, neutrophils not only proliferated but were activated, which demonstrated that neutrophils may participate in the onset of KD. After IVIG treatment, the activity of neutrophils was inhibited in some patients, however, in patients with CALs, neutrophils were constantly activated, suggesting that neutrophils inhibition may be one of the mechanisms of IVIG. And neutrophils may play a valuable role in the development of coronary artery lesions in Kawasaki disease.