抑制Ras 信号通路可减弱小鼠胚胎干细胞的造血分化_医学论文.docVIP

抑制Ras 信号通路可减弱小鼠胚胎干细胞的造血分化_医学论文 抑制Ras 信号通路可减弱小鼠胚胎干细胞的造血分化_医学论文 作者：王晓燕　刘兵　要晖宇　侯宁　杨晓　于晓妉　毛宁【摘要】 　　为了探讨Ras信号通路对小鼠胚胎干细胞(Embryonic Stem cells, ES cells)造血分化的影响，将突变型基因RasN17转染小鼠ES细胞，免疫印迹检测RasN17的表达对Erk1/2及Akt磷酸化的作用，应用半定量RTPCR检测ES细胞在分化过程中与造血相关的基因的表达。结果表明：Ras N17的表达能抑制Erk1/2和Akt的磷酸化，携带RasN17基因的ES细胞在分化过程中Runx1 、SCL 及betamajor珠蛋白等基因的表达被显著抑制， 而FLK1的表达不受影响。结论：ES细胞体外造血分化需要Ras通路的活化。 【关键词】 胚胎干细胞； 造血分化； Ras 　　 Suppression of Ras Mediated Signaling Attenuates Hematopoietic Differentiation of Embryonic Stem Cells of Mice In Vitro Abstract To investigate the possible involvement of Ras signaling in the hematopoietic differentiation of embryonic stem cells (ES cells), ES cells were transfected with RasN17, the dominantnegative mutant of Ras. Western blot was used to test the effect of RasN17 expression on Erk1/2 and Akt phosphorylation, semiquantitative RTPCR was used to detect expression of gene related to hemalopoiesis in differentiation of ES cells. The results showed that the expression of RasN17 in the ES cells remarkably downregulated the phosphorylation of Erk1/2 and Akt simultaneously. Moreover, the expression of several markers related with hematopoiesis including Runx1, SCL and betamajor globin， were significantly suppressed in the EB expressing RasN17, whereas the transcription of Flk1, a gene required earlier than SCL in development of hematopoietic and endothelial lineages, was not influenced. It is concluded that the activation of Ras is pivotal for in vitro hematopoietic differentiation of ES cells. Key words embryonic stem cell hematopoiesis differentiation Ras Ras是由1条多肽链组成的低分子量蛋白，由原癌基因ras编码而命名，包括K，N，H 3种类型。Ras的活性取决于其与GTP及GDP的结合，与前者的结合是其活化形式。 它是MAPK(Erk1/2)及PI3K等信号通路上游的重要组成成分，并通过参与调控这些信号通路而影响细胞的增殖与分化。最近研究发现，Ras及其下游信号分子均参与调控小鼠的胚胎发育，并且不同类型的Ras对胚胎发育特别是造血发育发挥不同的调控作用［1］。胚胎干细胞(embryonic stem cell, ES cell)是一种具有全能分化特性的细胞，其体外造血分化可以模拟并重现体内胚胎造血过程。报道表明：Ras及其下游的信号通路对ES细胞体外培养过程中全能性的维持非常重要［2］。但Ras蛋白能否调控小鼠ES细胞体外造血分化并不明确。为此，本研究探讨了Ras信号通路阻断对ES细胞造血分化的影响。 材料和方法 材料 DMEM、IMD