Liquid Culture Model For Adult Hematopoiesis At Molecula

我们之前对造血干细胞/祖细胞的研究表明，在EPO或G-CSF刺激的独特培养系统下，固定的红系细胞能够转化为髓系细胞，反之亦然。特定细胞的表型可能通过细胞发育不同阶段表达的基因和蛋白质的模式来反映。了解这两种谱系的可能机制？目前，我们将这项研究扩展到E14、G14、E14和E14？G14和G14？培养系统内的E14细胞。细胞群研究更重要，因为它们更准确地反映了细胞持续相互作用的生理现实。采用RAGE法共检测了266个表达的基因特异性片段，其中65%（171个）的基因产物在D0、E14和G14内共享，3%~9%的基因在14 d点被观察到个体群体的特征，并分为11种不同的表达模式。共同表达的基因暗示了红系和髓系谱系之间的密切发育关系，并为这两个谱系提供了基础。相互转换。145个（55%）cDNA产物已被鉴定为已知的细胞周期调节因子、转录因子和谱系特异性基因。先前鉴定的145个片段中有27个（18%）功能未知，这些基因可能与出现在同一生物过程中的已知基因共同作用，3个（2%）被定义为新序列。利用2DE技术获得的蛋白点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中细胞内蛋白的定性和定量表达谱。数据表明有一些细胞群专门负责细胞因子？EPO和G-CSF的信号通路，并表现出某些细胞同时负责EPO和G-CSF的分子现象，但确切的是相互矛盾而非协同的生物学效应，这表明确实发生了基因重编程，对这两个因子的基础研究和治疗目的具有重要的指导意义。目前，EPO或G-CSF在原代和二代培养细胞中持续上调或下调的基因产物和蛋白点，以及这三个新基因已被选择进行进一步研究，并将在未来的文章中报道。这是首次在mRNA和蛋白水平上同时研究红细胞和髓细胞的谱系发育。研究表明，红系和髓系之间的发育关系比以前认为的要密切得多，基因重编程（谱系互转换）可能发生在这两个谱系的细胞分化和开关过程中。了解造血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.