Objective: To explore whether thrombopoietin (TPO) can rescue megakaryopoiesis by protecting bone marrowderived endothelial progenitor cells (BM-EPCs) in patients receiving chemotherapy for hematological malignancies.
Methods: Bone marrow samples were collected from 23 patients with hematological malignancies 30 days after chemotherapy and from 10 healthy volunteers. BM-EPCs isolated from the samples were identified by staining for CD34, CD309 and CD133, and their proliferation in response to treatment with TPO was assessed using CCK8 assay. DiL-Ac-LDL uptake and FITC-UEA-I binding assay were performed to evaluate the amount of BM-EPCs from the subjects. Tube-formation and migration experiments were used for functional assessment of the BM-EPCs. The BM-EPCs with or without TPO treatment were co-cultured with human megakaryocytes, and the proliferation of the megakaryocytes was detected with flow cytometry.
Results: Flow cytometry indicated that the TPO-treated cells had high expressions of CD34, CD133, and CD309. CCK8 assay demonstrated that TPO treatment enhanced the proliferation of the BM-EPCs, and the optimal concentration of TPO was 100 μg/L. Double immunofluorescence assay indicated that the number of BM-EPC was significantly higher in TPO-treated group than in the control group. The TPO-treated BM-EPCs exhibited stronger tube-formation and migration abilities (P < 0.05) and more significantly enhanced the proliferation of co-cultured human megakaryocytes than the control cells (P < 0.05).
Conclusions: TPO can directly stimulate megakaryopoiesis and reduce hemorrhage via protecting the function of BM-EPCs in patients following chemotherapy for hematological malignancies.
目的: 探索血小板生成素（TPO）能否通过修复大剂量化疗后患者的骨髓内皮祖细胞（BM-EPC）恢复其对巨核细胞的造血支持作用。
方法: 选取23例血液系统恶性肿瘤患者化疗后30 d的骨髓以及10例健康人骨髓作为实验标本。采用表面特异性抗原CD34，CD309，CD133对BM-EPC进行鉴定；使用CCK8分析TPO是否可促进血液肿瘤患者化疗后BM-EPC增殖及其最佳作用浓度。设置TPO处理组为实验组，无TPO为对照组，健康人BM-EPC为健康对照组；通过DiL-Ac-LDL摄取及FITC-UEA-I结合实验检测实验组、对照组和健康对照组的BM-EPC数量。利用成管及迁移实验评估3组的BM-EPC功能；分别将实验组和对照组BM-EPC与巨核细胞共培养，利用流式细胞术检测巨核细胞增殖情况。
结果: 实验组BM-EPC高表达CD34/CD133/ CD309；TPO可促进BM-EPC增殖，最佳作用浓度为100 μg/L。免疫荧光双标实验显示，实验组BM-EPC数量较对照组明显增加（P＜0.05）。实验组成管能力及迁移能力较对照组增强（P＜0.05）。共培养后，实验组巨核细胞数多于对照组（P=0.013）。
结论: 大剂量化疗后患者BM-EPC受损，TPO具有直接刺激巨核系统造血作用，还可能通过促进BM-EPC增殖，修复其功能，从而恢复BM-EPC对巨核细胞的造血支持作用。
Keywords: endothelial progenitor cells; megakaryopoiesis; thrombopoietin.