Liquid Culture Model For Adult Hematopoiesis At Molecula

我们之前对造血干/祖细胞的研究表明，在EPO或G-CSF刺激的独特培养系统下，承诺的红系能够转换为髓系细胞，反之亦然。特定细胞的表型可能通过在细胞发育的不同阶段表达的基因和蛋白质的模式来反映。为了了解S分化和转换这两个谱系的可能机制，我们目前将这一研究扩展到E14、G14、E14？G14和G14？E14五个细胞群体的红系和髓系发育过程中的分子事件。细胞群体研究更有意义，因为它们更准确地反映了细胞持续相互作用的生理现实。利用RAGE方法分析了266个基因特异表达片段，其中65%(171个)的基因产物在D0、E14和G14之间共享，3%~9%的基因在14日龄时具有个体特征，可分为11种不同类型的表达模式。共同表达的基因暗示了红系和髓系之间密切的发育关系，以及这两个谱系的基础？彼此皈依。145个(55%)的基因产物与已知的细胞周期调控基因、转录因子和谱系特异性基因相一致。在145个已鉴定的未知功能基因片段中，27个(18%)可能与同一生物过程中出现的已知基因协同功能，3个(2%)被定义为新序列。利用2DE技术得到的蛋白质点的表达谱与细胞群体的基因表达分布模式相似。我们比较了五种细胞群体中细胞内蛋白的定性和定量表达谱。提示存在一些细胞因子--S信号转导的细胞群，并呈现出某些细胞同时负责EPO和G-CSF的分子现象，但它们的生物学效应是完全相反的，而不是协同作用的，这表明确实发生了基因重排，对基础研究和治疗目的都有重要的指导意义。目前，在原代和继代培养细胞中用EPO或G-CSF持续上调或下调的基因产物和蛋白质点，以及这三个新基因已被选作进一步研究，并将在未来的文章中报道。这是首次同时在mRNA和蛋白质水平上研究红系和髓系细胞的谱系发育。这项工作表明，红系和髓系之间的发育关系比以前认为的要密切得多，在这两个谱系的细胞分化和转换过程中，可能发生了基因重新编程(谱系相互转换)。了解造血细胞分化和转换的机制将带来有价值的见解。

Our previous work of hematopoetic stem/progenitor cells has shown that committed erythroid lineage is capable of switching into myeloid lineage cells, and vice versa, under a unique culture system with EPO or G-CSF stimulation. The phenotype of specific cells might be reflected by patterns of genes and proteins expressed in various stages of cellular development. To understand the possible mechanisms of these two lineage?s differentiation and switches, we have currently extended this study into the molecular events during the process of erythroid and myeloid development with five cell populations of E14, G14, E14?G14 and G14?E14 cells within the culture system. Cell population studies are more significant since they more accurately reflect the physiologic reality of continuously interacting cells. Using RAGE method, a total of 266 expressed gene-specific fragments were investigated, of which 65% (171) of total gene products were shared within D0, E14, and G14, 3%~9% of genes were observed characteristic for individual populations at 14-day point, and grouped into 11 different types of expression patterns. The co-expressed genes implicate a close development relationship between erythroid and myeloid lineages and a basis for these two lineages? conversion each other. 145 (55%) cDNA products have been identified with known genes of cell cycle regulators, transcription factors, and lineage specific genes. 27 (18%) of 145 fragments previously identified genes with unknown function, these genes may co-function with the known genes that appear in same biological process, and 3 (2%) were defined as novel sequences. The expression profile of protein spots using 2DE technique shows similar patterns to the gene expression distribution of cell populations. We have compared qualitative and quantitative expression profiles of intracellular proteins among the five cell populations. The data suggest there are some cell populations of being responsible specifically for cytokine?s signaling and exhibit a molecular phenomena that some cells are responsible for both EPO and G-CSF at same time, but exact contradict, rather than synergic, biological effect, which indicates a true gene reprogram happened and an important guidance for both basic study and therapeutic purpose with these two factors. Currently, gene products and protein spots, which were consistently up-regulated or down-regulated in primary and secondary culture cells with EPO or G-CSF, as well as the three novel genes have been chosen for further study and will be reported in future articles. This is the first time that the lineage development of erythroid and myeloid cells has been studied simultaneously at both mRNA and protein levels. The work indicates that there is a much more close development relationship between erythroid and myeloid lineages than thought before, and gene reprogramming (lineage interconversion) may take place during these two lineage cellular differentiation and switches. Understanding mechanisms of hematopoietic differentiation and switch will lead to valuable insights.