Analysis on the mechanisms of growth, differentiation, and survival of megakaryocytic cells

1.为了探讨Flt3TKD突变的激活机制，我们通过构建一系列小鼠Flt3TKD突变体(MFlt3Asp838Val)中所有22个胞质酪氨酸残基的单一Tyr→Phe替换突变体(MFlt3Asp838Val)，对突变体的结构性激活和下游信号转导的关键酪氨酸残基进行了分析。Tyr845Phe、Tyr892Phe和Tyr922Phe替换抑制了mFlt3Asp838Val自身的磷酸化，抑制了ERK1/2、STAT3和STAT5的激活，抑制了细胞的非因子依赖性增殖和存活。相反，这三个Tyr→Phe突变部分抑制了野生型Flt3的配体依赖的激活和抗凋亡活性，表明这些酪氨酸残基对mFlt3Asp838Val.的结构性激活和信号转导更为关键。2.我们检测了Notch和HOXB4诱导的造血干细胞自我更新过程中细胞周期调节分子的表达，发现这两个分子都诱导c-myc的表达。…此外，我们还确定HOXB4通过-195~-161碱基之间的元件激活c-My启动子。我们还证明，在适当的细胞因子存在的情况下，利用Myc/ERT单独诱导c-myc活性足以促进造血干细胞的自我更新，这表明4-羟基他莫昔芬对c-myc活性有反应。这些结果表明，Notch和HOXB4通过诱导c-myc来诱导造血干细胞的自我更新。Anamorsin^&lt；-/-&gt；小鼠是胚胎致死的，原因是胎肝中明确的造血失败，尽管这些小鼠胎肝中的造血干/祖细胞数量并没有减少，但髓系，特别是红系集落的形成严重中断。此外，Anamorsin^&lt；-/-&gt；红系细胞在终末成熟过程中启动了细胞凋亡。至于Anamorsin介导的细胞存活机制，基因芯片分析显示，Anamorsin^-lt；-/-&gt；小鼠胎肝中Bclxl和JAK2的表达严重受损。较少

1.To explore activation mechanism of FLT3 TKD mutation, we analysed critical tyrosine residues for the constitutive activation and downstream signaling of the mutant by generating a series of single Tyr→Phe substitution mutant of all 22 cytoplasmic tyrosine residues of murine FLT3 TKD-mutant (mFLT3Asp838Val). Tyr845Phe, Tyr892Phe and Tyr922Phe substitutions suppressed the phosphorylation of mFLT3Asp838Val itself, the activation of Erk1/2,STAT3 and STAT5, and the factor-independent cell proliferation and survival. In contrast, these three Tyr→Phe mutations partially suppressed but maintained the ligand-dependent activation and anti-apoptotic activity of wild-type FLT3, suggesting that these tyrosine residues were more critical for the constitutive activation and signaling of mFLT3Asp838Val.2.We examined the expression of cell cycle regulatory molecules during Notch- and HOXB4-induced self-renewal of hematopoietic stem cells, and found that both molecules induce the expression of c-myc. … More In addition, we determined that HOXB4 activated the c-my promoter through the element between -195 and -161 bp. We also proved that the induction of c-myc activity alone was sufficient for enhancing self-renewal of hematopoietic stem cells in the presence of appropriate cytokines using Myc/ERT, which reveals c-myc activity in response to 4-hydroxytamoxifen. These results indicated that Notch and HOXB4 induce self-renewal of hematopoietic stem cells through the induction of c-myc.3.We generated knock out mice for Anamorsin. Anamorsin^<-/-> mice are embryonic lethal due to the failure of definitive hematopoiesis in the fetal liver, Although the number of hematopoietic stem/progenitor cells in the fetal liver did not decrease in these mice, myeloid and particularly erythroid colony formation was severely disrupted. Also, Anamorsin^<-/-> erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the fetal liver of Anamorsin^<-/-> mice. Less