儿童免疫性血小板减少症中自噬上调抑制前血小板形成

Enhanced Autophagy Suppresses Proplatelet Formation in Pediatric Immune Thrombocytopenia

目的:研究儿童免疫性血小板减少症(ITP)中巨核细胞自噬水平上调对前血小板生成的影响。方法:通过吉姆萨染色和免疫荧光染色法分别观察巨核细胞形态及前血小板的生成,Western blot法检测细胞骨架蛋白和自噬相关蛋白的表达,自噬调节药物(自噬诱导剂Rap,自噬抑制剂3-MA)调控小鼠巨核细胞的自噬。结果:ITP患儿巨核细胞内存在空泡状结构,自噬相关蛋白LC3-II/I和复合物Atg12-Atg5的表达量均显著高于对照组(LC3-II/I:ITP 1.32±0.18 vs Ctrl 0.49±0.16,P<0.05;Atg12-Atg5:ITP 0.69±0.17 vs Ctrl 0.12±0.08,P<0.05);免疫荧光染色显示,ITP患儿巨核细胞细胞微丝排布异常,同时肌球蛋白磷酸化显著增强((ITP 0.74±0.09;Ctrl 0.05±0.02,P<0.05);自噬诱导剂和抑制剂能调控小鼠巨核细胞生成前血小板并改变细胞周期蛋白的表达,Cyclin D1(Veh 1.08±0.12;Rap 0.46±0.04;Rap+3-MA 0.70±0.03),Cyclin D2(Veh 0.47±0.04;Rap 0.27±0.04;Rap+3-MA 0.41±0.03),P21(Veh 0.15±0.01;Rap 0.04±0.01;Rap+3-MA 0.05±0.01)。结论:ITP患儿巨核细胞自噬上调是前血小板形成障碍的关键因素。

Objective:To investigate the effect of enhanced autophagy in megakaryocyte to proplatelet formation in children with immune thrombocytopenia(ITP).Methods:Giemsa staining and immunofluorescence staining were used to observe megakaryocyte morphology and proplatelet formation,Western blot was used to determine the expression of cytoskeleton protein and autophagy related protein.Autophagr regulation drugs Rap or 3-MA was used to regulate autophagy of megakaryocytes.Results:Some vacuole-like structures was found in ITP megakaryocytes of the children,the expression of LC3Ⅱ/Ⅰ(ITP 1.32±0.18;Ctrl 0.49±0.16 P<0.05)and Atg5-Atg12(ITP 0.69±0.17;Ctrl 0.12±0.08,P<0.05)was significantly higher in ITP children as compared with those in control group.The immu-nofluorescence staining showed that the cytoskeleton arrangement in megakaryocytes of ITP children was abnormal,and the phosphorylation of myosin light chain was also increased(ITP 0.74±0.09,Ctrl 0.05±0.02,P<0.05).In vitro,inducer or inhibitor of autophagy could regulate the production of proplatelet and the expression of cell cycle related protein,including CyclinD1(Veh 1.08±0.12;Rap 0.46±0.04;Rap+3-M A 0.70±0.03),CyclinD2(Veh 0.47±0.04;Rap 0.27±0.04;Rap+3-MA 0.41±0.03),P21(Veh 0.15±0.01;Rap 0.04±0.01;Rap+3-M A 0.05±0.01).Conclusion:Enhanced autophagy is the key factor of poor proplatelet formation in megakaryocytes of ITP children.