Animal Models of Polycythemia Vera

本研究的长期目标是明确真性红细胞增多症(PV)的分子基础。 PV通过对承诺的造血祖细胞的作用代表了突变的激酶JAK2 V617F的最终表型，但我们假设虽然JAK2 V617F的表达与PV的临床表型有关，但导致疾病的潜在分子机制存在于 不需要JAK2的造血干细胞(HSC)和血小板生成素(TPO)受体MPL参与了这些机制。这一假设是基于我们发现PV患者MPL表达受损并伴随着血浆TPO增加，以及我们观察到这一缺失 在JAK2V617F转基因(TG)小鼠的PV模型中，MPL基因的缺失消除了PV的表型，为从干细胞水平理解PV的病理生理学提供了机制基础，并为此提供了一种合理的治疗方法。 MPL：TPO轴，以及在人PV CD34细胞中上调的特定基因在PV小鼠JAK2 V617F TG模型中对HSC行为的作用。在特定的目标1中，我们将检测TPO基因的去除对JAK2 V617F TG小鼠的表型、其HSC池大小和HSC的影响 基因表达谱，通过与TPO-/-小鼠繁殖。对照实验将使用MPL功能被点突变或与MPL无关的基因敲除而取消的小鼠，而中和MPL抗血清将作为靶向治疗的模型进行检验。具体而言 目的2，我们将建立一个表达PV MPL剪接变异体的TG小鼠，并在JAK2 V617F存在和不存在的情况下评估其表型。我们还通过杂交的方法，检测了剔除在PV中上调的两个基因SPARC或Lcn2对JAK2V617F TG小鼠表型的影响。 最后，在特定的目标3中，我们将使用NOG小鼠的异种移植来检查来自临床上不同的PV患者群体的基因定义的PV CD34细胞的体内行为，这些患者群体是我们通过基因表达谱和无监督的层级聚类确定的。

The long-temri objective of this research project is to define the molecular basis of polycythemia vera (PV). PV represents the ultimate phenotype of the mutant kinase, JAK2 V617F, through its effects on committed hematopoietic progenitor cells but we hypothesize that while JAK2 V617F expression is responsible for the clinical phenotype of PV, the underlying molecular mechanisms responsible for the disease reside in the hematopoietic stem cell (HSC), which does not require JAK2, and that the thrombopoietin (TPO) receptor, Mpl, is integrally Involved In these mechanisms. This hypothesis, which is based on our discovery of impaired Mpl expression in PV with an attendant increase in plasma TPO, and our observation that absence of the MPL gene abrogated the PV phenotype in a JAK2 V617F transgenic (tg) mouse model of PV, provides a mechanistic basis for understanding the pathophysiology of PV at the stem cell level and a rational approach to therapy To this end, we propose to use genetic techniques to dissect the influence of the MPL:TPO axis, and also the roles of specific genes up regulated in human PV CD34+ cells, on the behavior of HSC in the murine JAK2 V617F tg model of PV. In Specific Aim 1, we will examine the effect of abrogation of the TPO gene on the phenotype of the JAK2 V617F tg mouse, on the size of its HSC pool and the HSC gene expression profile, by breeding with a TPO-/- mouse. Control experiments will employ mice in which Mpl function was abrogated by a point mutation or a gene knockout independent of MPL that impairs platelet production, while a neutralizing Mpl antiserum will be examined as a model of targeted therapy. In Specific Aim 2, we will create a tg mouse expressing a PV Mpl splice variant and assess its phenotype in the presence and absence of JAK2 V617F. We also examine by crossbreeding, the effect of knocking out SPARC or LCN2, two genes that are up regulated in PV, on the phenotype of the JAK2 V617F tg mouse. Finally, in Specific Aim 3, we will use xenotranslantation in NOG mice to examine the in vivo behavior of genetically-defined PV CD34 + cells from clinically distinct PV patient populations that we have identified by gene expression profiling and unsuoervised hierarchical clustering.