Stk40参与小鼠造血系统发育和谱系分化的功能和机制研究

Our previous study demonstrated that Stk40, a putative serine/threonine kinase, could promote mouse embryonic stem cell differentiation and be pivotal for mouse embryonic development. Recently, we found that embryos of Stk40 knockout mice, compared to control mice, had a significantly reduced size of hematopoietic organs, including fetal liver and spleen, suffering from moderate anemia with reduced circulating blood cell numbers and evident subcutaneous edema. Moreover,the percentages of monocytes (Mac1+/Gr1low)and granulocytes (Mac1+/Gr1high) were significantly higher in the spleen and fetal liver of Stk40 knock out embryos than in that of control embryos. These observations indicate that Stk40 may be a novel regulator important for the fetal hematopoiesis. However, how Stk40 controls the embryonic hematopoiesis and whether it is also associated with the hematopoiesis in the adult remain unknown. Thus, in this project, we plan to address the following three issues:1) the role of Stk40 in mouse fetal liver hematopoietic stem cell(HSC) homeostasis and lineage differentiation; 2) the role of Stk40 in mouse bone marrow HSC homeostasis and lineage differentiation; 3) the molecular mechanism by which Stk40 functions in the regulation of hematopoiesis. This study will elucidate the functions of Stk40 in hematopoiesis and help understanding the regulation of hematipoiesis networks.

Stk40是一个新发现的具有丝苏氨酸激酶结构域的蛋白。本课题组过去的研究表明Stk40对小鼠胚胎干细胞的分化和小鼠胚胎的正常发育与新生呼吸具有重要的作用。我们的前期研究表明，Stk40基因敲除小鼠胎肝、脾脏体积显著减小；循环血细胞数量明显减少,敲除小鼠出现贫血并伴随严重的皮下水肿。此外，胎肝和脾脏中的单核细胞和粒细胞比例显著增加。这些发现提示Stk40对胚胎发育阶段造血系统的发育和分化可能有重要的作用。但是，我们尚不清楚Stk40如何调节胚胎造血系统的发育和是否参与成体造血系统的调节。在本项目中，我们拟研究：1）Stk40在小鼠胚胎造血干细胞调控和血细胞分化中的作用；2)Stk40在成体造血干细胞调控和血细胞谱系分化中的作用；3）Stk40影响造血系统发育和造血干细胞分化的分子机制。这些研究对深入了解Stk40在造血系统中的功能和造血干细胞分化调控网络具有重要意义。

Stk40（Serine/threonine kinase 40）是一个含有丝氨酸苏氨酸激酶结构域并在进化化上高度保守的蛋白。本课题组的研究结果表明，在小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）中Stk40位于全能因子Oct4的下游并激活Erk/MAPK信号通路并诱导其向胚外内胚层(extraembryonic endoderm, ExEn)分化。关于Stk40全基因敲除小鼠(conventional knock out)的研究表明，小鼠在出生前后死亡，体表苍白，肺泡发育异常。除此之外，我们的研究还揭示Stk40具有抑制脂肪细胞分化和促进骨骼肌细胞分化的功能。为了全面了解Stk40基因的生理功能，本课题研究了Stk40在小鼠胎肝红系细胞定向分化过程中的作用和机制。.红系细胞的分化过程受到一系列细胞外信号分子和细胞内转录因子及信号通路的调控。我们的研究揭示Stk40参与调控小鼠胎肝红系造血(definitive erythropoiesis)的过程。缺失Stk40导致小鼠胎肝发育不良，细胞数目减少，凋亡细胞增加以及红系细胞分化出现部分阻滞。体外集落形成细胞(colony forming cell, CFC)检测显示，胎肝中所有含红系的集落数目均显著性减少；流式分选的巨核系-红系祖细胞(megakaryocyte-erythrocyte progenitor, MEP)在体外集落培养中，形成的集落数目亦减少。造血干祖细胞(hematopoietic stem and progenitor cell, HSPC)所占的细胞比例无明显改变，均可以实现多系分化与长期重建受体小鼠的血液系统与免疫系统。除此之外，缺失Stk40的胎肝成红细胞在体外形成成红细胞造血岛(erythroblastic island, EBI)的能力减弱。.小鼠胚胎14.5天(embryonic day, E14.5)的胎肝芯片数据显示缺失Stk40后出现肿瘤坏死因子（tumor necrosis factor-α, TNF-α）的异常激活，IκB蛋白水平下降以及TNF-α通路相关基因的上调表达。缺失Stk40后出现部分无效造血(dyserythropoiesis)很有可能是由于TNF-α通路的激活，剪切的Caspase-3增加与Gata1减少引起的。