| Abstract: | 兒童慢性免疫性血小板低下症的致病機轉主要是由於抗血小板抗體所導致的血小板破壞，而在動物的模式顯示這種血小板抗體會引發血小板凋亡。近年的報告也指出在血小板疾病中，具調控細胞凋亡路徑的CXCR4表現量有下降的情形。本研究主要目的在評估是否慢性免疫性血小板低下症的孩童身上血小板凋亡的現象以及CXCR4的表現是否受到影響。本研究針對21病人和12對照組進行檢查。使用流式細胞儀(flowcytometry)，檢驗血小板凋亡的指標包括annexin V, caspase 3和粒腺體跨膜電位去極化(mitochondria inner transmembrane potential depolarization)。結果發現慢性免疫性血小板低下症的孩童其血小板呈現細胞凋亡陽性的比率減少。CXCR4在病人組的表現有較高的現象；利用流式細胞儀分析結果與對照組比較 P=0.001，利用西方墨點轉漬法(western blotting)分析結果與對照組比較 P=0.013。結果同時顯示CXCR4的下游蛋白質，磷酸化Akt在病人組的表現有較高的現象。綜合以上結果，慢性免疫性血小板低下症的孩童，發生血小板凋亡抗性，這種現象可能與CXCR4表現增強與Akt的活化相關。
進一步檢測慢性免疫性血小板低下孩童血漿中與輔助性T細胞相關之細胞激素表現，將慢性兒童免疫性血小板低下症分類為疾病活化期(血小板數值小於<50 × 109/l)，穩定期(50 到150 × 109/l)和緩解期(> 150 × 109/l)，探討第一型、第二型、第三型和第十七型輔助性T細胞之細胞激素的表現是否有不同。招募並抽取57位慢性免疫性血小板低下孩童和28位健康對照組的週邊血液，利用酵素結合免疫吸附分析法(ELISA)分析血漿中INF-γ, IL-2, IL-4, IL-10,TGF-β1和 IL-17等細胞激素。實驗結果顯示IL-17和 IFN-γ的血漿濃度在慢性免疫性血小板低下的疾病活動期和穩定期有顯著的上升，並且呈現正向相關(r = 0.640, P < 0.001)。TGF-β1的表現在疾病的活動期(P < 0.001)或穩定期(P=0.001)，皆明顯地下降；並且與血小板數值呈現負向相關(r = 0.355, P = 0.007)。本研究意謂IL-17和 IFN-γ的上升在慢性免疫性血小板低下症之兒童扮演重要的角色;疾病的活性則與TGF-β1的表現下降有相關。
The pathogenesis of childhood chronic primary immune thrombocytopenia (ITP) is mainly mediated by antiplatelet autoantibodies, which have been shown to induce platelet apoptosis in murine models. Decreased CXCR4 expression, which can regulate apoptotic pathway, has been described in platelet disorders. The study aims to determine whether platelet apoptosis is increased in pediatric patients with chronic ITP and whether there is any involvement of the CXCR4 chemokines axis. Twenty-one patients and 12 controls were studied. Using flow cytometry, we investigated apoptotic markers of platelets including annexin V, caspase 3, and mitochondrial inner transmembrane potential depolarization. The percentage of the platelets with apoptosis-positive markers was not increased in chronic ITP patients. CXCR4 expression was higher in the patients as detected by flow cytometric (P=0.001) and western blotting analysis (P=0.013). The results also revealed that CXCR4 downstream proteins, Akt phosphorylation was more frequent in chronic ITP patients than controls. The study shows platelet apoptosis resistance existing in pediatric patients with chronic ITP. It may be associated with enhanced CXCR4 expression and Akt activation.
The study further investigated whether plasma levels of Th cytokines in pediatric patients with chronic ITP were different compared with healthy controls. Fifty-seven pediatric patients with chronic ITP and 28 healthy controls were enrolled. Patients were divided into three groups based on their platelet counts at the time of the study, (i) active disease < 50 × 109 /l (n=23), (ii) stable disease 50-150 × 109 /l (n=23), and (iii) in remission > 150 × 109 /l (n=11). Plasma concentration of Th1 [interferon gamma (INF-γ), interleukin 2 (IL-2)], Th2 (IL-4, IL-10), Th3 [transforming growth factor-β1 (TGF-β1)], and Th17 (IL-17) cytokines were investigated by enzyme-linked immunosorbent assay. There was a positive correlation between IL-17 and IFN-γ levels in chronic ITP patients (r= 0.640, P<0.001). Reduced TGF-β1 expression was observed in patients with active (P < 0.001) and stable disease (P = 0.001) in comparison with controls. Moreover, TGF-β1 level in patients was positively correlated with the platelet count (r= 0.355, P=0.007). Elevation of IL-17 and IFN-γ may be an important dysregulation of cellular immunity in pediatric patients with chronic ITP. The disease activity is associated with reduced production of TGF-β1. |
