Relationship between the expression of complement regulatory proteins and PIG-A gene abnormalities in CD34^+ cells of PNH.

应用流式细胞仪双色分析技术检测了阵发性睡眠性血红蛋白尿症(PNH)患者和健康志愿者的红细胞、血小板、粒细胞、红细胞、T、B淋巴细胞和CD34~+细胞表面糖基磷脂酰肌醇(GPI)锚定的膜蛋白CD55和CD59的表达。此外，我们还研究了患者粒细胞、T淋巴细胞和CD34~+细胞来源的磷脂酰肌醇糖链A类(PIG-A)基因的异常。结果如下：1.我们发现PNH患者多种细胞CD55和CD59的表达是不一致的。特别是，我们研究了患者循环红细胞表型和培养的红细胞表型之间的关系。结果表明，所有患者培养的红细胞均由PNH I、II、III红细胞或PNH II、III红细胞组成，但仍有成熟红细胞的最终表型。这些发现…更多的S认为，PNH中红细胞的最终表型是通过PNH II红细胞的消失或持续来决定从红细胞到红细胞的成熟过程(Shichishima等人，血液，在出版社)。采用聚合酶链式反应和逆转录聚合酶链式反应检测PNH患者外周血粒细胞和T淋巴细胞中PIG-A基因的异常，并进行核苷酸序列分析。我们的研究重点是用流式细胞仪检测这些细胞的表型与PIG-A基因异常克隆的比例之间的关系。我们的结果提示，PNH患者粒细胞的表型不是由PIG-A基因异常决定的，而是GPI-core的大小决定的，尽管PNH的发生与PIG-A基因的缺陷有关(Noji，Shichishima等人，血液86：132a，1995)。单色流式细胞仪分析显示，健康志愿者骨髓CD34~+细胞为单一的CD59阳性细胞，而PNH患者的CD34~+细胞为CD59阳性细胞。此外，我们还发现PNH患者骨髓CD34^+细胞中PIG-A基因异常与PNH患者外周血粒细胞中PIG-A基因异常相同。PNH患者外周血CD34~+细胞中是否存在上述两种PIG-A基因异常有待进一步研究。较少

We investigated the expression of glycosylphosphatidylinositol (GPI)-anchored membrane proteins (CD55 and CD59) by erythrocytes, platelets, granulocytes, erythroblasts, T and B lymphocytes, and CD34^+ cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) and healthy volunteers using flow cytometric two-color analysis. In addition, we also studied the abnormality of phosphatidylinositol glycan-class A (PIG-A) gene derived from granulocytes, Tlymphocytes, and CD34^+ cells in the patients. The results are as follows :1. We found that the expression of CD55 and CD59 by various cells as described above was discordant in PNH patients. In particular, we studied relationship between the phenotypes of circulating erythrocytes and cultured erythroblasts in the patients. Our results showed that the cultured erythroblasts in all the patients consisted of PNH I,II,and III erythroblasts or PNH II and III erythroblasts in spite of the final phenotype of mature erythrocytes. These finding … More s suggest that the final phenotype of erythrocytes in PNH is determined during maturation from erythroblasts to erythrocytes by the disappearance or persistence of PNH II erythroblasts (Shichishima et al., Blood, In press).2. We investigated PIG-A gene abnormalities in peripheral blood granulocytes and T lymphocytes obtained from PNH patients by PCR and RT-PCR followed by nucleotide sequence analysis. Our present study focused on the relationship between the phenotypes of these cells detected by flow cytometric analysis and proportion of abnormal clone of the PIG-A gene. Our results suggest that PIG-A gene abnormalities can not prescribe the phenotypes of granulocytes from PNH patients but the volume of GPI-core, although a defect of PIG-A gene certainly underlies the occurence of PNH (Noji, Shichishima et al., Blood 86 : 132a, 1995).3. One-color flow cytometric analysis showed that the CD34^+ cells derived from bone marrow blood from healthy volunteers consisted of a single population positive for CD59, while those from PNH patients had a continuous population from negative to positive for CD59. Moreover, we found that PIG-A gene abnormalities in bone marrow blood CD34^+ cells were same as those in peripheral blood granulocytes obtained from patients with PNH.We will have to evaluate carefully whether the CD34^+ cells could have the above two abnormalities of PIG-A gene in PNH. Less