血小板生成素可体外修复原发性免疫性血小板减少症患者受损的内皮细胞

Effect and mechanism of thrombopoietin on repairing ITP vascular microenvironment in vitro

目的研究血小板生成素(TPO)对原发性免疫性血小板减少症(ITP)患者内皮祖细胞(EPC)的保护作用。方法从ITP患者及健康供者标本分离BM-EPCs,将研究对象分为3组:ITP/+TPO组、ITP/-TPO组及正常对照。CCK8法检测TPO对BM-EPC的促增殖作用,流式细胞仪标记小鼠抗人CD133、小鼠抗人CD34和小鼠抗人血管内皮生长因子受体(CD309)单克隆抗体的共表达用以鉴定BM-EPC,免疫荧光实验DiL-Ac-LDL和FITC-UEA-I检测EPCs的数量,成管、迁移实验及BM-EPC与巨核细胞共培养检测BM-EPCs的功能。结果分离的BM-EPCs培养5 d后呈不规则形、椭圆形,培养7~10 d后呈典型长梭形样。CCK-8检测结果显示,TPO促进EPCs增殖,最佳药物浓度为100 ng/mL。流式细胞仪检测各组BM-EPC,结果显示高表达CD34/CD133/CD309,免疫荧光双染细胞数高于ITP/-TPO组(n=8,P<0.05)。与ITP/-TPO组相比,ITP/+TPO组成管能力增强(n=12,P<0.05),共培养巨核细胞数量增多。结论 TPO对ITP患者受损的内皮细胞具有修复作用,主要表现为成管功能及巨核细胞支持功能明显增强。

Objective To investigate the protective effect of TPO on EPCs of ITP patients. Methods BM-EPCs were isolated from ITP patients and healthy donor specimens. The experiment cases was divided into 3 groups: ITP/+TPO group, ITP/-TPO group and normal control. CCK8 was used to explore the proliferative effect of TPO on BM-EPCs. BM-EPCs was identified by flow cytometry with co-expression of mouse anti-humanCD133, mouse anti-human CD34 and mouse anti-human vascular endothelial growth factor receptor(CD309) monoclonal antibodies. Moreover, the number of EPCs was evaluated with DiL-Ac-LDL uptake and FITC-UEA-I binding assay. Furthermore, the function of BM-EPCs was explored by using tubeformation, BM-EPC and MK co-culture experiments. Results The separated BM-EPCs exhibited irregular shape and elliptical shape after 5 days and a typical longer fusiform after 7-10 days. TPO promoted the proliferation of EPCs with a optional drug concentration of 100 ng/ml. The number of cells with immunofluorescence double staining in TPO group was significantly higher than of control group(n=8,P<0.05). The tube formation(n=12) and the number of co-cultured MK were significantly enhanced in TPO-treated group(P<0.05). Conclusion TPO may exert megakaryopoiesis effect in ITP patients via protection of BM-EPCs. The main manifestation is that the tube formation function and the megakaryocyte support function are significantly enhanced.