摘要
骨髓增生性疾病(Myeloproliferative disorders,MPDs)是指骨髓组织持续增殖而引起的一组疾病,常表现为造血干细胞(包括红细胞、粒细胞、巨核细胞和血小板)的一系或多系细胞恶性增生。真性红细胞增多症(Polycythemia vera,PV)是一种以红细胞增多为主要特征的MPDs。PV患者造血祖细胞在体外培养时对许多生长因子和细胞因子高度敏感且细胞中蛋白质酪氨酸磷酸化异常,但是这些细胞表面的生长因子和细胞因子受体并无异常,表明这些细胞内的PTKs和PTPs可能发生了改变。通过对候选PTPs和PTKs编码区cDNA的测序分析,在24例PV患者中我们发现20例患者JAK2存在G:C→T:A点突变。该突变呈现多样的杂合现象,即突变的比率是随机的,并不符合孟德尔遗传定律,这表明此突变是后天获得性的突变而非先天遗传因素所致。突变直接导致其编码的JAK2激酶也发生了改变,也就是JAK2激酶的假激酶结构域——JH2区域的617位上的缬氨酸残基(V)被苯丙氨酸残基(F)所取代。JAK2V617F突变体激酶活力明显升高,当JAK2V617F与EPOR在Hela细胞中共同表达时,其可持续激活EPO诱导的信号通路。这一功能获得性突变解释了PV患者造血祖细胞对许多生长因子和细胞因子高度敏感的原因。为了证明JAK2V617F是导致PV的原因我们还成功地在小鼠中表达了hJAK2V617F基因,得到可生存并繁殖后代的转基因小鼠模型。而JAK2激酶突变体的高水平表达,使小鼠表现出一系列与MPDs非常相似的表型特征,这说明在小鼠中,JAK2V617F能导致MPDs的发生,同时我们也为研究JAK2V617F的病理学作用以及开发治疗MPDs的方法和药物提供了优秀的动物模型。
Myeloproliferative disorders are a group of conditions that cause an overproduction of blood cells -- platelets, white blood cells, and red blood cells -- in the bone marrow. Though myeloproliferative disorders are serious, and may pose particular health risks, individuals with these conditions often live for many years after diagnosis. Myeloproliferative disorders include: Polycythemia vera (PV), Essential thrombocytosis (ET), Primary myelofibrosis (PMF) and Chronic myelogenous leukemia (CML).
Polycythemia vera (PV) is a human clonal hematological disorder. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C to T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val617 to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.
To clarify JAK2V617F is the cause of PV, we have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2V617F, showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2V617F expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2V617F can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2V617F and to develop treatment for MPDs.
引文
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