流式细胞术分析ITP巨核细胞分化发育异常的初步研究

Study on dysmegakaryocytopoiesis of patients with idiopathic thrombocytopenic purpura by using flow cytometry

目的建立流式细胞术(FCM)分析人骨髓巨核细胞的新方法,用于特发性血小板减少性紫癜(ITP)巨核细胞分化发育异常的初步研究。方法对15例正常骨髓供者(对照组)、37例ITP患者及11例继发性血小板减少症(ST)患者进行骨髓巨核细胞绝对计数;采用Percoll密度梯度分离法富集骨髓巨核细胞,经透膜处理后,用荧光素标记的单克隆抗体标记巨核细胞糖蛋白Ⅱb/Ⅲa(CD41a)、血管性血友病因子(vWF)、P-选择素(CD62P),用碘化丙锭(PI)标记巨核细胞DNA,流式细胞术分析巨核细胞倍体分布和糖蛋白表达水平。结果对照、ITP和ST三组之间比较,骨髓巨核细胞绝对数量无差异(P>0.05)。富集后的骨髓标本,巨核细胞比例显著增高,可比富集前高17倍,而且DNA倍体(N)分布清晰,易于FCM分析设门和计算。与对照组比较,ITP患者巨核细胞DNA倍体分布未见明显改变,但CD41a表达量在2N、4N、8N、16N、32N、64N显著高于对照组(P<0.01);vWF表达量在各倍体巨核细胞均减低,32N时减低更显著(P<0.05);CD62P表达量在ITP组与对照和ST组间差异无显著性(P>0.05)。结论在ITP患者骨髓巨核细胞的分化发育过程中,其数量和DNA倍体增加未受影响,但其功能蛋白合成出现显著变化。在不同倍体巨核细胞中,CD41a表达显著增高;而vWF呈现表达下降趋势,尤其是高倍体(32N)时减低更为显著;P-选择素表达水平稳定。多色流式细胞术分析骨髓巨核细胞为ITP的发病机制及其相关研究提供了新手段。

Objective To setup a flow cytometric measurement of human bone marrow megakaryocyte which is used to investigate the dysmegakaryocytopoiesis of patients with idiopathic thrombocytopenic purpura （ITP）. Methods Bone marrow megakaryocytes were absolutely counted in 15 normal bone marrow donors （normal group）, 37 patients with ITP and 11 patients with secondary thrornbocytopenia （ST）. Marrow cells were initially enriched for megakaryocytes by a PereoU density gradient. After permeabilizd, the megakaryocytes were labeled with a fluorescent monoclonal antibodies against to the GPIIb/IIIa complex（ CD41a）, von Willebrand factor （vWF） and P-seleetin（CD62P）, then stained with propidium iodide （PI） for DNA quantitation. Megakaryocytes were analyzed by flow cytometry for DNA ploidy distribution and glycoprotein expression. Results The absolute number of megakaryocytes from human bone marrow aspirates was no significant difference among the normal group, ITP group and ST group （ P 〉 0.05）. The frequency of megakaryocytes in fractionated bone marrow was enhanced 17-fold enrichment and DNA ploidy distribution was more distinct compared with unfractionated bone marrow, thus it is easy to gate and calculate for FCM analysis. In contrast with normal group, DNA ploidy distribution of patients with ITP was no significant change and the expression level of megakaryocyte CD41a in patients with 1TP significantly increased from 2N to 64N （ P 〈 0.01 ）. At the same time, vWF expression of megakaryocyte decreased in each ploidy, especially significantly in 32N （p〈 0.05）. There was no significant difference of CD62P expression among patients with ITP, normal controls and ST group （ P 〉 0.135）. Conclusion： During the human marrow megakaryocyte differentiation and maturation of patients with ITP, the megakaryocyte number and DNA ploidy in patients with ITP were not affected. However, the synthesis of the function protein has significantly changed. During the all ploidy stages of patients with ITP, megakaryocyte expressed the increased CD41a level, while vWF expression show declined trend, especially in the higher ploidy（32N）, its expression decreased significantly. CD62P was expressed constantly during all ploidy. Multicolor flow cytometric analysis for bone marrow megakaryocyte provide a new mean for researching the ITP mechanism.