A Liquid Culture System Model For Adult Hematopoiesis

造血谱系分化的基因表达模式的生物学意义还知之甚少。在这项研究中，我们检测了丰富的人CD133+(干/祖)细胞的红系和髓系的基因表达模式和表达基因的比较分析。从5个细胞群体中提取细胞总RNA，反转录成cDNA，用320个针对血液发育基因序列的特异引物进行RAGE(快速分析基因表达)聚合酶链式反应。聚合酶链式反应产物经8%TBE凝胶分离，检索GeneSystem 320TM数据库或DNA测序鉴定。表达的266个基因特异片段的mRNA表达模式可分为3组(11种类型)：(1)在单细胞群体中特异表达的基因(I、III型)，(2)在2个细胞群体中表达的基因(IV、VI型)，(3)在3个或3个以上群体中表达的基因(类型VIII、XI型)。在145个已定义的cDNA中，有3个(2%)是新基因。用双向凝胶电泳法测定的同一群体的蛋白质图谱与重叠和区分的基因图谱非常一致。流式细胞术还检测到谱系特异性抗原在谱系承诺过程中的共表达。具体地说，基于CD13(髓系)和CD36(红系)表达的细胞分选表明这些谱系中存在CD13和CD36双阳性细胞。进一步的克隆性分析显示，在EPO、G-CSF或EPO+G-CSF的作用下，CD13+/CD36+的CD13-/CD36-细胞组不能诱导出红系爆裂和集落形成单位(BFU-E和CFU-E)、粒细胞集落形成单位(CFU-G)和混合集落(CFU-GE)。另一方面，CD13或CD36单阳性群体细胞几乎只产生CFU-G或CFU-E，而不产生含有上述细胞因子的CFU-GE。此外，本实验还观察了G-CSF对单纯髓系祖细胞的作用以及EPO对红系和髓系祖细胞的影响。本研究提示CD13+/CD36+细胞即使在单个细胞因子中培养4周后仍具有分化为髓系和红系的潜能。这些数据支持一种假设，即正常造血祖细胞上的谱系限制性抗原的共表达为应激反应中的谱系可塑性提供了一种机制。用GoSurfer程序对红系和髓系表达的基因进行比较分析，发现两者的生物学过程有显著差异，表明两个谱系共有的基因主要参与发育、刺激反应和信号转导途径，而两者各自特异表达的基因主要参与生物过程的调节和细胞程序性死亡。我们的结论是，谱系转换可能是正常造血的特征，祖细胞上谱系特异性抗原的共表达可能为基因表达重叠和谱系可塑性提供基础。

The biological implications of gene expression patterns of hematopoietic lineage differentiation is poorly understood. In this study, we examined gene expression patterns and comparative analysis of expressed genes in enriched erythroid and myeloid lineages of human CD133+(stem/progenitor) cells. Total cellular RNA was extracted form 5 cell population pellets, reverse transcribed into cDNA, and subjected to RAGE (rapid-analysis-gene-expression) PCR amplification using 320 primers specific for gene sequences of blood development. PCR products were separated through 8% TBE gel and identified by searching the GeneSystem 320TM database or DNA sequencing. mRNA expression patterns of expressed 266 gene-specific fragments were categorized into 3 groups (11 types): (1) genes expressed specifically in a single cell population (Types I?III), (2) genes expressed in 2 cell populations (Types IV?VII), and (3) genes expressed in 3 or more populations (Types VIII?XI). Of 145 defined cDNAs, 3 (2%) were novel genes. Protein profiles of same populations determined by 2-dimensional gel electrophoresis were in good agreement with overlapped and distinguished gene patterns. Flow cytometry also detected the co-expression of lineage-specific antigens during lineage commitment. Specifically, cell sorting based on CD13 (myeloid) and CD36( erythroid) expression demonstrated the existence of double-positive CD13 and CD36 cells in these lineages. Further clonagenic analysis showed that erythroid burst- and colony-forming units (BFU-E and CFU-E), granulocyte colony-forming units (CFU-G), and mixed colonies (CFU-GE) were induced in CD13+/CD36+, but not in CD13-/CD36- cell fractions with EPO, G-CSF, or EPO plus G-CSF. On the other hand, single-positive CD13 or CD36 population cells generated almost exclusively CFU-G or CFU-E, but no CFU-GE with above cytokines. In addition, the effect of G-CSF on myeloid only and EPO on both erythoid and myeloid progenitors was observed through the experiment. The study suggests that CD13+/CD36+ cells possess the potential for differentiation of myeloid and erythroid lineages even after 4-week culture in a single cytokine. These data support the hypothesis that co-expression of lineage-restrictive antigens on normal hematopoietic progenitors provides a mechanism for lineage plasticity in response to stress. Comparative analysis of genes expressed in erythroid and myeloid lineages using GoSurfer program showed statistically significant differential biological processes and indicated that genes shared in both lineages involved mainly in development, response to stimulus, and signal transduction pathways, while genes specifically expressed in either alone mostly in regulation of biological process and programmed cell death. We conclude that lineage conversion may be a characteristic of normal hematopoiesis, and the co-expression of lineage-specific antigens on progenitors may provide the basis for gene expression overlap and lineage plasticity.