STAT1与FAXDC2在巨核细胞生成中的功能和机制研究

发布时间：2018-05-21 21:20

本文选题：MPL + W515L　；参考：《武汉大学》2016年博士论文

【摘要】：血小板是血液系统的重要组成部分,在机体止血中发挥着重要的功能。巨核细胞是血小板形成的前体细胞,巨核细胞的发育分化和血小板的形成,受到多种转录因子和信号通路的共同调控。巨核细胞的过度增殖以及发育分化异常,导致骨髓增殖性肿瘤及急性巨核细胞白血病的发生。研究巨核细胞分化的分子机制,不仅能够加深对造血细胞发育分化的理解,也能促进对相关疾病的认识,为疾病为疾病的治疗提供科学依据和潜在靶标。持续激活型血小板生成素受体MPL突变(MPLW515L)诱发PMF病人的脾脏和骨髓中产生大量异常发育的巨核细胞,导致原发性骨髓纤维化。MPLW515L持续激活多条信号通路,包括J AK/STATs、PI3K/AKT和MAPK/ERK。研究表明血小板生成素主要激活STAT3和STAT5,也能激活STAT1.STAT1信号转导在正常的巨核细胞分化中起着重要的功能,但其分子机制和在原发性骨髓纤维化中的作用尚未被完全阐述。为了探究STAT1在原发性骨髓纤维化发病机理中的功能,我们首先检测MPLW515L激活的下游信号通路的变化,结果显示磷酸化的STAT3和STAT5显著上升,但STAT1的总蛋白和701位酪氨酸的磷酸化的STAT1都显著降低,另一巨核细胞分化关键蛋白GATA1也显著降低,同时细胞凋亡的比例减少,但是细胞周期并无明显变化。这些数据提示STAT1可能在MPLW515L介导的异常巨核细胞分化与增殖的信号通路中与凋亡和GATA1表达相关。另一方面,我们发现RUNX1启动子区域内存在保守性的STAT1的结合位点。通过双荧光素酶实验与染色质免疫沉淀技术,我们证实STAT1通过结合RUNX1启动子调控RUNX1的表达。同时,我们发现显性抑制性的RUNX1能够一部分抑制STAT1介导的巨核细胞分化。这些证据显示STAT1可能是通过调控RUNX1实现对巨核细胞生成的调控。最后,我们在STAT1基因敲除小鼠的骨髓细胞中过表达MPLW515L建立体内原发性骨髓纤维化小鼠模型。我们发现STAT1基因敲除后明显加快了疾病的进程：白细胞和血小板数异常增多的更加明显、骨髓纤维化加重和异常巨核细胞增殖更加严重。这些证据说明STAT1信号转导的异常是导致原发性骨髓纤维化中异常巨核细胞生成的关键因素。巨核细胞白血病是一类罕见的白血病,表现为巨核细胞的无限增殖,但丧失终末分化导致血小板低下。巨核细胞白血病的治疗缺乏有效的靶标,常规放化疗预后非常差,亟需开发新的治疗方法。已有研究表明,分化治疗是巨核细胞白血病治疗的有效手段。鉴定和研究巨核细胞分化中的功能基因,可以为分化治疗提供潜在的治疗靶标。FAXDC2是脂肪酸羟化酶家族中的成员,脂肪合成在巨核分化中发挥重要功能。因此,我们对FAXDC2基因在巨核分化中的功能进行了深入研究。首先,我们发现FAXDC2的表达量伴随巨核细胞分化的过程而显著上升；与之形成显著差异的是FAXDC2的表达量在急性髓系白血病和急性巨核细胞白血病中显著下降。其次,过表达FAXDC2促进小鼠原代细胞向巨核细胞分化。以上结果说明,FAXDC2是巨核细胞分化的过程中的正调控因子。分子机制的研究结果显示过表达FAXDC2促进了转录因子RUNX1的表达。并且,FAXDC2过表达拯救了由显性抑制RUNX1引起的巨核细胞分化受阻。最后,过表达FAXDC2导致鞘脂类合成酶GM3的上升,这提示我们FAXDC2可能通过促进脂类代谢促进巨核细胞分化。本研究阐释了STAT1基因在原发性骨髓纤维化中的作用及其分子机制,结果显示STAT1表达下调是MPLW515诱发骨髓纤维化的重要因素。同时,本论文探索了FAXDC2在巨核细胞分化中的功能及其与急性巨核细胞白血病的潜在联系,研究表明FAXDC2是促进巨核细胞分化的重要因素,其异常低表达可能与白血病的形成相关。我们的研究进一步加深了对原发性骨髓纤维化和巨核细胞白血病这一类与巨核细胞异常分化相关的疾病,为此类疾病的靶向治疗提供新的思路。
[Abstract]:Platelets, an important part of the blood system, play an important role in the hemostasis of the body. Megakaryocytes are precursors of platelets. The development and differentiation of megakaryocytes and the formation of platelets are regulated by a variety of transcription factors and signaling pathways. The molecular mechanism of megakaryocyte differentiation can not only deepen the understanding of the development and differentiation of hematopoietic cells, but also promote the understanding of the related diseases, and provide a scientific basis and potential target for the treatment of disease. The continuous activated thrombopoietin receptor MPL process MPLW515L induced a large number of abnormal development of megakaryocytes in the spleen and bone marrow of PMF patients, resulting in the continuous activation of multiple signal pathways in primary myelofibrosis.MPLW515L, including J AK/STATs, PI3K/AKT and MAPK/ERK. studies indicating that thrombopoietin mainly activates STAT3 and STAT5, and activates STAT1.STAT1 signal transduction in normal. Megakaryocyte differentiation plays an important role, but its molecular mechanism and role in primary myelofibrosis have not been fully elucidated. In order to explore the function of STAT1 in the pathogenesis of primary myelofibrosis, we first detected the changes in the downstream signaling pathways activated by MPLW515L. The results showed that the phosphorylated STAT3 and STAT5 were significant. However, the total protein of STAT1 and the STAT1 phosphorylation of 701 tyrosine decreased significantly, and the other megakaryocyte differentiation key protein GATA1 decreased significantly, while the proportion of cell apoptosis decreased, but the cell cycle did not change significantly. These data suggest that STAT1 may differentiate and proliferate in MPLW515L mediated abnormal megakaryocytes. The signaling pathway is associated with apoptosis and GATA1 expression. On the other hand, we have found that there is a conserved STAT1 binding site in the RUNX1 promoter region. By double luciferase experiment and chromatin immunoprecipitation technique, we confirm that STAT1 regulates RUNX1 by combining the RUNX1 promoter. At the same time, we find the dominant inhibitory RUNX1 energy. Enough to inhibit STAT1 mediated megakaryocyte differentiation. These evidence suggests that STAT1 may be regulated by the regulation of RUNX1 for megakaryocyte formation. Finally, we have expressed MPLW515L in the bone marrow cells of the STAT1 knockout mice to establish a mouse model of primary myelofibrosis. We found that the STAT1 gene knockout is obvious. The progression of the disease is that the abnormal number of leukocytes and platelets is more obvious, myelofibrosis is aggravated and abnormal megakaryocyte proliferation is more serious. These evidence suggests that abnormal STAT1 signal transduction is a key factor in the formation of abnormal megakaryocyte in primary myelofibrosis. Megakaryocyte leukemia is a rare class. Leukaemia is characterized by the unlimited proliferation of megakaryocytes, but loss of terminal differentiation leads to thrombocytopenia. The treatment of megakaryocyte leukemia lacks an effective target. The prognosis of conventional radiotherapy and chemotherapy is very poor, and new therapies are urgently needed. The study has shown that differentiation therapy is an effective means of megakaryocyte leukemia. Functional genes in the differentiation of nuclear cells, which can provide a potential therapeutic target for differentiation therapy,.FAXDC2 is a member of the fatty acid hydroxylase family, and fat synthesis plays an important role in megakaryocyte differentiation. Therefore, we have studied the function of the FAXDC2 gene in megakaryocyte differentiation. First, we found that the expression of FAXDC2 is associated with it. The process of megakaryocyte differentiation was significantly increased, and the significant difference between the expression of FAXDC2 in acute myeloid leukemia and acute megakaryocytic leukemia was significant. Secondly, overexpression of FAXDC2 promoted the differentiation of murine primary cells to megakaryocytes. The above results indicated that FAXDC2 was a positive modulation in the process of megakaryocyte differentiation. The results of molecular mechanism study showed that overexpression of FAXDC2 promoted the expression of transcription factor RUNX1. And FAXDC2 overexpression saved the differentiation of megakaryocyte caused by dominant inhibition of RUNX1. Finally, overexpression of FAXDC2 led to the rise of sphingolipid synthetase GM3, which suggests that FAXDC2 may promote the megaky by promoting lipid metabolism. This study explains the role of STAT1 gene in primary myelofibrosis and its molecular mechanism. The results show that the downregulation of STAT1 expression is an important factor in MPLW515 induced bone marrow fibrosis. At the same time, this paper explores the function of FAXDC2 in megakaryocyte differentiation and the potential association with acute megakaryocyte leukemia. The study shows that FAXDC2 is an important factor in promoting megakaryocyte differentiation, and its abnormal low expression may be associated with the formation of leukemia. Our study further deepens the disease related to the abnormal differentiation of megakaryocyte, such as primary myelofibrosis and megakaryocyte leukemia, and provides a new way of thinking for targeting therapy of such diseases.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R551.3

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