脐血CD34+细胞体外培养生成成熟巨核细胞并产出血小板的研究

目的：诱导脐血造血细胞在体外分化为成熟的巨核细胞并产出血小板，以探讨血小板的生成及释放机制。方法：通过采用优化的培养体系对免疫磁珠分选后得到的脐血CD34^＋细胞分别进行向巨核系分化的液体培养和集落培养，将培养的细胞和分离的上清液中的血小板样颗粒进行流式细胞术、免疫组织化学染色、光镜、电镜及血小板聚集实验的检测。结果：培养的巨核细胞有前血小板伸出，培养的巨核细胞和血小板样颗粒与正常的巨核细胞和血小板结构一致。免疫组织化学染色检测显示培养的细胞表达血小板特异性抗原GPⅡbⅢa的阳性率为95％以上，且为强阳性。培养的血小板大小颗粒与正常血小板均能对凝血酶产生聚集反应。流式细胞仪检测显示培养的血小板与正常血小板均具有同样的CD41高表达率。结论：脐血造血细胞能在体外诱导生成高纯度且成熟的巨核细胞并产出血小板．为血小板生成机制研究提供一个良好的技术平台。

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OBJECTIVE: To induce hematopoietic progenitor/stem cells of umbilical cord blood to differentiate into mature megakaryocytes and platelets in vitro and to investigate the mechanism of production of platelets. METHODS: The CD34+ cells were sorted from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in vitro with optimized medium to be differentiated into mature megakaryocytes and platelets. The cultured cells and the platelet-like particles were isolated from the culture and were checked by the fluorescence-activated cell sorter (FACS), immunohistochemistry assays, light microscope,electron microscope and platelet aggregation tests. RESULTS: The cultured megakaryocytes were detected with proplatelets and both the cultured cells and the platelet-sized particles were found to have the same structure with the normal megakaryocytes and platelets by light and electron microscope. The immunohistochemistry assays revealed the cultured cells expressed GP II b III a with a positivity of 95% which was a special antigen for platelets and megakaryocytes. Culture-derived platelet-sized particles aggregated in response to thrombin as the plasma derived-platelets did. The cultured platelets had the same positivity of CD41 as the platelets from platelet rich plasma. CONCLUSION: The hematopoietic progenitor/stem cells can be induced to differentiate into purified and mature megakaryocytes and platelets. It provides a practical way to study the mechanism of platelets production.