造血调控分子PPP2Cβ参与红系分化及EDAG在白血病发生中的作用研究

发布时间：2017-12-27 15:11

本文关键词：造血调控分子PPP2Cβ参与红系分化及EDAG在白血病发生中的作用研究　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

更多相关文章： PPP2Cβ 红系分化 EDAG 白血病

【摘要】：红细胞的生成受到多层次、多水平的调控,其中转录因子在红细胞生成过程中发挥重要的调控作用。GATA1是目前研究最为深入的红系转录调控因子。GATA1一方面可通过翻译后水平修饰如乙酰化、磷酸化等,进而调控其自身转录活性,另一方面还可与其它多种转录因子形成动态复合体,对下游基因进行选择性调控,进而参与红系分化。本研究主要围绕GATA1的两个相互作用蛋白PPP2C?与EDAG展开研究,揭示了PPP2C?与GATA1相互作用并调控红系分化的新功能,并探讨了EDAG在BCR-ABL及MLL-AF9诱导的白血病中的作用。本研究分为以下两部分:第一部分:PPP2C?与GATA1相互作用促进红系分化的研究我们实验室前期利用酵母双杂交构建了造血干细胞(HSC)转录因子相互作用网络,在该网络中,蛋白磷酸酶2A的催化亚基β(PPP2Cβ)是GATA1新的相互作用蛋白,提示PPP2Cβ很可能调控造血分化。本部分我们首先利用促红细胞生成素(Erythropoietin,EPO)诱导脐血CD34+细胞向红系分化,检测了PPP2Cβ在分化过程中的表达,发现无论是在m RNA水平还是蛋白水平PPP2Cβ都发生明显上调,而在分化晚期恢复正常水平,表明PPP2Cβ很可能在红系早期分化过程中发挥重要功能。进一步我们构建了PPP2Cβ带Flag标签的表达载体,利用免疫共沉淀实验证实GATA1和PPP2Cβ存在相互作用,并确定了PPP2Cβ结合在GATA1的N端锌指结构。利用双荧光素酶报告基因实验证明,PPP2Cβ能增强GATA1的转录调控活性且能促进GATA1下游靶基因的转录。进一步利用慢病毒表达系统,构建过表达PPP2Cβ的K562细胞稳定株,发现PPP2Cβ过表达可促使K562细胞自发向红系分化。这些结果表明PPP2Cβ可调控GATA1活性,是一种新的红系分化调控分子。第二部分:EDAG在白血病发生中的作用研究红系分化相关基因(Erythroid Differentiation Associated Gene,EDAG)是我们实验室率先分离克隆的新基因,特异表达于造血组织,参与调控造血细胞的增殖、分化与HSC的谱系分化平衡。我们前期研究发现EDAG可与GATA1相互作用,通过募集p300增强GATA1乙酰化水平及转录调控活性,三者形成动态复合体,选择性激活红系分化相关基因,进而促进红系分化。同时,我们也发现EDAG除了促进红系分化外,还可能与白血病的发生有关。本部分我们探讨了EDAG在BCR-ABL诱发的CML(慢性髓系白血病)及MLL-AF9诱发的AML(急性粒细胞白血病)发生中的作用。我们首先对BCR-ABL逆转录病毒进行了包装,获得高滴度病毒,并制备了BCR-ABL诱发的CML小鼠模型。对BCR-ABL感染的脐血CD34+细胞进行检测发现EDAG表达显著上调。进一步将BCR-ABL病毒感染EDAG基因敲除小鼠和正常小鼠的骨髓细胞,进行骨髓移植。结果显示敲低EDAG可降低BCR-ABL诱发的CML发病率,表现为移植EDAG敲除小鼠骨髓细胞的受体小鼠发病时间明显晚于对照小鼠,外周血白细胞数目显著低于对照组,这些结果表明EDAG在BCR-ABL诱发的CML发病过程中发挥重要作用。此外,我们利用MLL-AF9转基因小鼠评价了EDAG敲除在AML细胞浸润中的作用。在发病的MLL-AF9小鼠骨髓及脾脏中,EDAG表达显著降低。进一步将发病的MLL-AF9转基因小鼠脾细胞移植到EDAG基因敲除小鼠和野生型小鼠体内,研究造血微环境对白血病细胞的影响,结果表明两组小鼠在血象各项指标中和外周血的谱系中并没有明显区别,且存活率没有差异。综上所述,我们揭示了PPP2Cβ是红系分化过程中一个新的调控因子;并发现EDAG在BCR-ABL逆转录病毒诱导的CML中发挥重要作用。
[Abstract]:The formation of red blood cells is regulated by multilevel and multilevel, and the transcription factors play an important role in the process of erythrocyte formation. GATA1 is the most deeply researched transcription factor in red system. On the one hand, GATA1 can regulate its transcriptional activity through translational horizontal modifications such as acetylation and phosphorylation. On the other hand, it can also form dynamic complexes with other transcription factors, selectively regulate downstream genes and participate in erythroid differentiation. In this study, we focused on the two interacting proteins of GATA1, PPP2C and EDAG, revealing the interaction between PPP2C and GATA1, and regulating the new functions of erythroid differentiation. We also discussed the role of EDAG in BCR-ABL and MLL-AF9 induced leukemia. This study is divided into two parts: the first part: PPP2C? And GATA1 interaction on promoting erythroid differentiation ourprevious using yeast two hybrid constructs of hematopoietic stem cells (HSC) transcription factor interaction network, in the network, protein phosphatase 2A catalytic subunit beta (PPP2C beta) is GATA1 the role of new proteins, suggesting that PPP2C might regulate hematopoietic differentiation of beta. In this part, we first use of erythropoietin (Erythropoietin, EPO) induced differentiation of umbilical cord blood red cell line CD34+, detect the expression of PPP2C beta in the differentiation process, it is found that significantly increase occurred in M RNA and protein levels of PPP2C beta, and returned to normal level in the late stage of differentiation, is likely to show that PPP2C beta play an important role in early erythroid differentiation. Further, we constructed the expression vector of PPP2C beta with Flag tag. The interaction between GATA1 and PPP2C beta was confirmed by CO immunoprecipitation assay, and the zinc finger structure at the N end of GATA1 was confirmed. The double luciferase reporter gene experiment showed that PPP2C beta could enhance the transcriptional regulation activity of GATA1 and promote the transcription of target gene in the downstream of GATA1. Further using lentiviral expression system, construct the over expression of PPP2C beta K562 cells showed that PPP2C overexpression, beta K562 cells can promote spontaneous erythroid differentiation. These results indicate that PPP2C beta can regulate the activity of GATA1 and is a new regulation molecule of red system differentiation. The second part: the role of erythroid differentiation related gene EDAG in the pathogenesis of leukemia (Erythroid Differentiation Associated Gene, EDAG) was first isolated in our laboratory, gene cloning, specific expression in hematopoietic tissue, involved in the regulation of hematopoietic cell proliferation, differentiation and HSC lineage differentiation balance. Our previous studies found that EDAG could interact with GATA1, enhance the level of GATA1 acetylation and transcriptional regulatory activity through recruitment of P300, and the three form dynamic complexes, which selectively activate erythroid differentiation related genes, and then promote erythroid differentiation. At the same time, we also found that EDAG may be associated with the development of leukaemia in addition to the promotion of red lineage differentiation. In this section, we explored the role of EDAG in the pathogenesis of BCR-ABL induced CML (chronic myeloid leukemia) and MLL-AF9 induced AML (acute myelocytic leukemia). We first packed the BCR-ABL retrovirus, obtained a high titer virus, and prepared a BCR-ABL induced CML mouse model. The detection of CD34+ cells from BCR-ABL infected umbilical cord blood found that the expression of EDAG was significantly up-regulated. The BCR-ABL virus was further infected with the EDAG gene knockout mice and the normal mice bone marrow cells, and the bone marrow transplantation was carried out. Results showed that knockdown of EDAG can reduce the incidence rate of CML induced by BCR-ABL, to transplant EDAG knockout onset time of recipient mice bone marrow cells of mice significantly later than control mice, peripheral white blood cell number was significantly lower than the control group, these results suggest that EDAG play an important role in the pathogenesis of BCR-ABL induced by CML process. In addition, we used MLL-AF9 transgenic mice to evaluate the role of EDAG knockout in the infiltration of AML cells. In the bone marrow and spleen of the MLL-AF9 mice, the expression of EDAG decreased significantly. The transplanted spleen cells of MLL-AF9 transgenic mice of EDAG gene knockout mice and the onset of wild type mice, effects of hematopoietic microenvironment of leukemia cells, the results showed that the two groups of mice on blood indicators and peripheral blood lineages were not significantly different, and the survival rate was no difference. In conclusion, we have revealed that PPP2C beta is a new regulatory factor in the process of erythroid differentiation, and it is found that EDAG plays an important role in BCR-ABL retroviral induced CML.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7

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