A Liquid Culture System Model For Adult Hematopoiesis At

造血谱系分化的基因表达模式的生物学意义尚不清楚。在这项研究中，我们检测了人类CD133+（干细胞/祖细胞）红细胞和髓系富集细胞系的基因表达模式和表达基因的比较分析。从5个细胞群小球中提取细胞总RNA，逆转录成cDNA，使用320种血液发育基因序列特异性引物进行RAGE（快速分析-基因表达）PCR扩增。PCR产物经8% TBE凝胶分离，通过检索GeneSystem 320TM数据库或DNA测序进行鉴定。266个基因特异性片段的mRNA表达模式可分为3组（11种类型）：(1)在单细胞群体中特异性表达的基因(I型？III),(2)在2个细胞群中表达的基因(IV型？(3)在3个或更多群体中表达的基因（VIII型？XI型）。在145个确定的cdna中，3个（2%）是新基因。二维凝胶电泳测定的同一群体的蛋白质谱与重叠和区分的基因模式一致。流式细胞术还检测了谱系特异性抗原在谱系承诺过程中的共表达。具体来说，基于CD13（髓系）和CD36（红系）表达的细胞分选表明，这些谱系中存在双阳性CD13和CD36细胞。进一步的克隆原分析表明，红细胞爆发和集落形成单位（BFU-E和CFU-E），粒细胞集落形成单位（CFU-G）和混合集落（CFU-GE）在CD13+/CD36+细胞中被诱导，但在含有EPO、G-CSF或EPO + G-CSF的CD13-/CD36-细胞中没有被诱导。另一方面，单阳性CD13或CD36群体细胞几乎只产生CFU-G或CFU-E，而不产生上述细胞因子的CFU-GE。此外，通过实验观察G-CSF仅对髓系细胞的影响，EPO对红细胞和髓系祖细胞的影响。该研究表明，CD13+/CD36+细胞即使在单一细胞因子中培养4周后也具有髓系和红系分化的潜力。这些数据支持了一种假设，即在正常造血祖细胞上共同表达谱系限制性抗原提供了一种应对应激的谱系可塑性机制。使用GoSurfer程序对红系和髓系中表达的基因进行比较分析，发现在统计学上存在显著差异的生物学过程，并表明两个谱系中共享的基因主要涉及发育、刺激反应和信号转导途径，而单独表达的基因主要参与生物过程和程序性细胞死亡的调节。我们得出结论，谱系转换可能是正常造血的一个特征，谱系特异性抗原在祖细胞上的共同表达可能为基因表达重叠和谱系可塑性提供了基础。

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.