IDENTIFICATION OF GENE EXPRESSION IN POLYCYTHEMIA VERA BY DIFFERENTIAL DISPLAY

真性红细胞增多症（PV）是一种以红细胞增多、粒细胞增多和血小板增多为特征的干细胞获得性克隆性造血疾病。在PV中有正常的红细胞生成素（EPO）依赖的红母细胞和一小部分异常的不依赖EPO的红祖细胞。这些异常的不依赖EPO的红系祖细胞增殖和分化为成熟红细胞的分子靶点尚不清楚。为了鉴定这两个红细胞群体中表达不同的基因，我们使用两相液体红细胞培养系统培养并分离了12例仅接受静脉切除率治疗的PV患者的EPO依赖性和EPO非依赖性红细胞。采用基于聚合酶链反应的差异显示法（DDRT-PCR）鉴定这两个亚群中差异表达的mrna。63个mRNA转录本在epo依赖组和独立组中表现出差异表达。利用反向点印迹法对这些mRNA转录本的表达强度进行确认和定量后，克隆了20个在epo非依赖性红系细胞中与epo依赖组相比表达下调或上调的转录本，并对其进行测序。这些转录物中有11个与已知序列具有高度同源性，包括B3 （β -珠蛋白基因），B7（铁蛋白重亚基mRNA）， A3 （Nrf1， nf - e2相关转录因子），B1（人nuc2同源物），D1（人翻译控制蛋白），并且在epo独立的红系细胞中下调，epo独立组的表达量分别比epo依赖组低26、19、18、11、7倍。据报道，cnc -碱性亮氨酸家族成员Nrf1 （A3）在胎儿肝脏造血过程中起重要作用；Nrf1突变纯合的小鼠由于胎儿肝脏造血异常而患贫血。据报道，酵母nuc2 （B1）的人类同源物以特定的方式与Rb蛋白相互作用。12例PV患者中A3、B1、D1的表达均通过Northern blot证实。A3， B1和D1在PV中的作用正在研究中。20个转录本中有9个与已知序列无同源性。9个未知转录本中有3个在epo独立组中上调。Northern blot分析证实了它们的表达和大小。利用人类cDNA文库获得了它们的完整cDNA序列。研究epo不依赖的红系细胞中下调和上调的cDNA转录本可能有助于了解PV的发病机制和可能的治疗靶点。

Polycythemia vera (PV) is an acquired clonal hematopoietic disorder of stem cells characterized by erythrocytosis, granulocytosis and thrombocytosis. In PV there are normal erythropoietin (EPO)-dependent erythroblasts and a fraction of abnormal EPO-independent erythroid progenitors. The molecular targets responsible for these abnormal EPO- independent erythroid progenitors to proliferate and differentiate into mature red cells are still unclear. To identify the genes expressed differently in these two populations of erythroid cells, we used a two phase liquid erythroid culture system to culture and separate the EPO- dependent and EPO-independent erythroid cells from 12 phlebotomy-only treated PV patients. The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed in these two subpopulation. Sixty-three mRNA transcripts display differential expression in EPO-dependent and independent groups. After using reverse dot blot to confirm and quantitate the intensity of these mRNA transcripts, twenty transcripts with downregulated or upregulated expression in EPO-independent erythroid cells compared with EPO-dependent group were cloned and sequenced. Eleven of these transcripts have high homology with already-known sequences, including B3 (beta-globin gene), B7 (ferritin heavy subunit mRNA), A3 ( Nrf1, NF-E2-related transcription factor), B1 (human nuc2 homologue), D1(human translationally controlled protein), and are downregulated in EPO-independent erythroid cells, with 26, 19, 18, 11, 7 fold lower expression in EPO-independent than in EPO-dependent groups, respectively. It has been reported that the CNC-basic leucine family member Nrf1 (A3) plays an essential role during fetal liver hematopoiesis; mice homozygous for the Nrf1 mutation suffer from anemia as a result of abnormal fetal liver hematopoiesis. The human homologue of yeast nuc2 (B1) has been reported to interacts with the Rb protein in a specific manner. The expression of A3, B1 and D1 in 12 PV patients was confirmed by Northern blot. The role of A3, B1 and D1 in PV is under investigation. Nine of twenty transcripts have no homology with known sequences. Three of nine unknown transcripts were upregulated in the EPO-independent group. By Northern blot analysis, their expression and size were confirmed. Their full cDNA sequence is being obtained by using a human cDNA library. Investigation of both downregulated and upregulated cDNA transcripts in EPO-independent erythroid cells may provide insights into the pathogenesis of and possible therapeutic targets in PV.