Objective: To explore the method for inducing the differentiation of bone marrow cells into megakaryocytes in vitro so as to use for evaluating the activity of traditional Chinese medicines.
Methods: The bone marrow cells were separated from femurs and tibias of mice. The experiments were divided into 4 groups: control (no adding cytokines), TPO (adding 50 ng/ml TPO), TPO+SCF (50 ng/ml+50 ng/ml) and TPO+SCF+IL-6+IL-9 (50 ng/ml+50 ng/ml+20 ng/ml+20 ng/ml). The bone marrow cells in 4 groups were cultured in vitro for 6 d. Then the cell growth status was observed by the inverted microscopy, and the cell count was detected by using the automatic cell counter. The ratio and absolute count of megakaryocytes were detected by flow cytometry.
Results: Compared with control, three induction methods could stimulate the differentiation of bone marrow cells into megakaryocytes in vitro. TPO could slightly enhance the differentiation of bone marrow cells into megakaryocytes. Both the combination of TPO and SCF, and the combination of TPO, SCF, IL-6 and IL-9 could intensively stimulate proliferation of bone morrow cells and promote the differentiation of bone marrow cells into megakaryocytes. The addition of IL-6 and IL-9 could decrease the proliferation of non-megakaryocytes, but promote the differentiation of bone marrow cells into megakaryocytes.
Conclusion: The optimized differentiation of bone marrow cells into megakaryocytes has been completed by co-induction regimen of TPO, SCF, IL-6 and IL-9, which can be used to screen and evaluate traditional Chinese medicines promoting formation of platelets.
题目: 体外诱导骨髓细胞分化成巨核细胞的方法学研究.
目的: 探讨体外将小鼠骨髓细胞诱导分化为巨核细胞的方案,用于中药活性评价.
方法: 从小鼠股骨和胫骨取骨髓细胞，在血小板生成素（TPO）及TPO和干细胞因子（SCF）或TPO、SCF、白介素-6（IL-6）和白介素-9（IL-9）联合诱导下，体外培养6 d。然后用倒置显微镜观察培养体系中的细胞生长情况，自动细胞计数仪检测细胞数量，流式细胞术分析培养体系中巨核细胞比例，并计算巨核细胞绝对数量.
结果: 单因子或多因子联合诱导方法均能体外促进骨髓细胞分化成巨核细胞，TPO轻微促进骨髓细胞分化成巨核细胞，TPO和SCF联合及TPO、SCF、IL-6和IL-9联合均显著促进骨髓细胞增殖和分化成巨核细胞，添加IL-6和IL-9可促进骨髓细胞分化成巨核细胞，减少非巨核细胞增殖.
结论: TPO、SCF、IL-6和IL-9联合具有最强的促进骨髓细胞分化为巨核细胞的作用，可以用于促血小板形成中药活性筛选和评价.