雌激素对放射损伤血小板减少症的救治作用及机制研究

发布时间：2018-01-23 00:39

本文关键词： 雌激素电离辐射血小板减少症巨核细胞多倍体形成 GATA1 血小板生成　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：随着科技的发展,核能及其相关的技术在军事、医学、工业等多个领域得到了更加广泛的应用。然而,核能为我们的生活带来便利的同时,也增加了由电离辐射引起放射损伤的潜在风险。尤其是核事故、核爆炸等情况下会产生大量的放射损伤伤员。此外,临床上接受放射治疗和骨髓移植的肿瘤患者也会受到一定程度的放射损伤。骨髓是放射损伤的主要靶器官。其中,放射损伤引起的血小板严重减少会导致机体出血和感染,甚至死亡。不幸的是,目前临床上血小板减少症的治疗药物种类较少,而且它们大多还存在着起效慢、副作用大、价格昂贵等缺点。因此,寻找一种有效、安全的放射损伤血小板减少症的预防和救治方法显得至关重要。血小板由骨髓中的巨核细胞产生,血小板生成过程主要包括造血干细胞定向分化为巨核祖细胞、巨核祖细胞大量扩增并分化为成熟巨核细胞、成熟巨核细胞释放血小板等多个阶段。其中巨核细胞的分化成熟是血小板产生的关键步骤。巨核细胞成熟首先进行核内有丝分裂以形成多倍体,然后巨核细胞进入一个胞质成熟过程,即脂质和蛋白质大量合成并形成分界膜系统,最后巨核细胞向血管窦伸出血小板前体并向血液循环中释放血小板。在体内,调控血小板生成最重要的细胞因子是血小板生成素(Thrombopoietin,TPO)。但是,我们及他人的研究发现体内还有其他因子也参与了血小板生成的调控。雌激素是哺乳动物体内非常重要的一种性激素,主要由卵巢产生和分泌,它在人体内有着广泛的生物学作用。多个研究报道,雌激素对造血系统也有影响作用。有趣的是,巨核细胞不但被证实自身能产生雌激素,而且雌激素和巨核细胞生成和血小板生成有一定的联系。大量的临床数据表明人体血小板水平也存在性别差异,而且这种性别差异与体内的雌激素水平有关。另外,大量的研究表明,雌激素不仅在体外能够促进巨核细胞分化及血小板前体形成,在体内也可促进血小板的产生,但是使用他莫昔芬或氟维司群抑制雌激素受体(estrogen receptor,ER)的激活则会抑制血小板的产生。这些研究表明雌激素参与了巨核细胞的生成和血小板的产生,但是雌激素作用于血小板生成的哪一阶段及其相关机制仍未完全阐明。雌激素是一种类固醇激素,主要通过雌激素受体ERα和ERβ发挥作用。ERα和ERβ是一类核受体,未激活时,ER位于细胞膜上,与配体雌激素结合后激活,从而形成同源二聚体(ERα/ERα或ERβ/ERβ)或异源二聚体(ERα/ERβ),然后由细胞膜移位到细胞核中,与靶基因启动子上游的雌激素反应元件(estrogen response element,ERE)结合而调节靶基因的表达,从而发挥其生物学效应。ERα和ERβ在全身表现为明显的组织特异性分布。ERα主要表达于性器官如卵巢、乳腺等,而ERβ主要表达于非性腺器官如骨髓、肺、结肠等。另外,ERα和ERβ在结构及功能如配体识别和结合、受体激活、辅活化子或辅阻遏物的募集、靶基因转录调节上也有明显的差异。已有研究报道,巨核细胞均表达ERα和ERβ,但是,哪种受体在雌激素对巨核细胞的作用中发挥主要作用及其下游的靶分子和信号转导通路仍有待证实。另一方面,雌激素是一种经典的抗放药物,在动物实验中都被证实有辐射保护作用,无论照前给药还是照后给药都可以显著提高存活率。同样在放疗病人照射前给予雌激素,可以减轻外周血白细胞下降的程度,但是雌激素能否用于预防和治疗放射损伤血小板减少症仍然缺乏相关的研究。围绕以上科学问题,本研究使用小鼠骨髓(bone marrow,BM)Sca1+细胞来源的原代巨核细胞、人脐带血(cord blood,CB)CD34+细胞来源的原代巨核细胞、人巨核祖细胞株M07e细胞、人成熟巨核细胞株Meg-01细胞及c-Mpl-/-和ERβ-/-敲除小鼠,首先利用正常的C57BL/6J小鼠建立亚致死量放射损伤血小板减少症模型,并在照射前或照射后给予雌激素处理,观察雌激素对血小板水平恢复的影响;再利用细胞活性分析、流式细胞术、细胞免疫荧光、激光共聚焦显微镜观察和血常规检测等技术分析雌激素对巨核细胞增殖、分化、多倍体形成、血小板前体形成及血小板产生的影响,以明确雌激素对血小板生成的调控作用及作用阶段;最后,再利用western blot、RT-PCR、q RT-PCR、小干扰RNA(si RNA)基因敲低、双荧光素酶报告系统、染色质免疫共沉淀(Ch IP)等方法,对雌激素促血小板生成作用的分子机制进行深入分析。主要研究结果和结论如下:1、通过建立小鼠急性放射损伤模型发现,急性放射损伤可导致小鼠血小板水平快速降低,而放射前雌激素预处理或放射后雌激素干预均能有效加速放射后小鼠血小板水平的恢复,缩短放射后血小板低谷期的持续时间,并显著提高受照后小鼠的生存率。说明传统抗辐射药物不但可以预防放射损伤血小板减少症,还对放射损伤血小板减少症有一定的救治作用。2、雌激素尽管不能促进巨核祖细胞的增殖,但却可以促进巨核细胞的分化成熟、多倍体形成、血小板前体的形成和血小板的产生,并且可以促进小鼠骨髓巨核细胞成熟分化,显著升高外周血血小板水平,说明雌激素可以促进巨核细胞晚期分化,从而促进血小板的生成。3、雌激素处理巨核细胞可以显著激活巨核细胞的ERβ受体,而非ERa受体,阻断或敲低ERβ可以显著抑制雌激素诱导的巨核细胞分化成熟和多倍体形成,提示ERβ可能是雌激素促血小板生成作用的主要靶受体。4、雌激素诱导巨核细胞ERβ激活后,在众多巨核细胞重要调控分子中发现GATA1的表达显著上调,而敲低ERβ几乎完全抑制了雌激素诱导的GATA1表达上调,提示GATA1可能是雌激素下游效应的主要靶分子。进一步通过双萤光素酶报告系统和染色质免疫共沉淀(Ch IP)实验证实ERβ可以和GATA1的启动子结合,可能的ERE序列位于-1380至-1574;另外,敲低巨核细胞GATA1的表达后,雌激素诱导巨核细胞多倍体形成效应消失。这些结果表明,GATA1是ERβ下游的靶分子,ERβ激活后可与GATA1启动子结合,从而诱导GATA1表达上调,进而促进巨核细胞多倍体形成。5、体外实验证明,雌激素还可诱导GATA1下游靶分子STAT1表达上调和活化增强;敲低巨核细胞ERβ和GATA1的表达后,雌激素并不能诱导STAT1表达上调和活化增强;另外,敲低STAT1的表达几乎可以完全抑制雌激素促多倍体形成的作用,提示STAT1可能是GATA1作用的下游靶分子,介导了雌激素促巨核细胞多倍体形成的作用。6、NF-E2是GAT1下游的一个靶分子,体外实验表明雌激素处理巨核细胞后可以诱导NF-E2的表达上调,而敲低GATA1则可完全抵消雌激素诱导的NF-E2表达上调;另外,NF-E2是巨核细胞血小板前体形成和血小板产生的关键调控分子,这些结果提示,NF-E2可能是GATA1作用的下游靶分子,介导了雌激素促血小板前体形成和血小板产生的作用。通过本实验研究,我们不仅揭示了雌激素在放射损伤血小板减少症中的预防和救治作用,而且明确了雌激素在血小板生成中的作用,深入阐明了雌激素促进巨核细胞多倍体形成和血小板产生的分子机制,从而为临床血小板减少症的预防和救治提供新思路、新方法。
[Abstract]:With the development of science and technology, nuclear energy and related technology in military, medical, industrial and other fields has been more widely used. However, nuclear power has brought convenience to our life, also increases the potential risk of radiation damage caused by ionizing radiation. Especially the nuclear accident, a large number of the wounded will produce radiation injury nuclear explosion case. In addition, the clinical cancer patients receiving radiation therapy and bone marrow transplantation may be affected by the radiation injury to a certain extent. The bone marrow is the main target organ of radiation damage. The radiation damage caused by severe will result in a reduction of platelet and infection, bleeding and even death. Unfortunately, the current clinical the treatment of thrombocytopenia fewer drugs, but most of them still exist slow onset, the side effect is big, expensive shortcomings. Therefore, finding an effective, safe radiation damage in blood The prevention and treatment method of plate to reduce the disease is crucial. Platelet megakaryocytes produced by bone marrow, platelet production process including hematopoietic stem cells to differentiate into megakaryocyte progenitor cells, megakaryocyte progenitor cells proliferate and differentiate into mature megakaryocytes, multiple stages of mature megakaryocyte differentiation. The release of platelet megakaryocyte maturation is a key step in platelet production. Megakaryocyte maturation first nuclear mitosis to form polyploid megakaryocytes, and then enter a cytoplasmic maturation process, namely lipid and protein synthesis and the formation of a large number of demarcation membrane system, finally to extend the megakaryocyte platelet precursors and vascular sinus to the blood platelet release. In vivo, regulation of platelet production of the most important factor is thrombopoietin (Thrombopoietin, TPO). However, the research found that the US and others There are other factors are also involved in the regulation of platelet production. Estrogen is a sex hormone in mammals is very important, mainly produced and secreted by the ovary, it has a wide range of biological effects in the body. Many research reports, estrogen also has an impact on the hematopoietic system. Interestingly, not only the megakaryocyte proven itself can produce estrogen, and have certain relation to estrogen and megakaryocytopoiesis and platelet production. A large number of clinical data show that human platelet level gender differences, but also related to the gender difference and estrogen level in the body. In addition, a number of studies have shown that estrogen can promote not only in vitro megakaryocyte differentiation and platelet precursors formed in vivo may also promote platelet production, but the use of tamoxifen or fulvestrant inhibition of estrogen receptor (estrogen rece Ptor, ER) activation can inhibit platelet production. These studies indicate that estrogen is involved in the formation of megakaryocyte and platelet production, but not what stage the action of estrogen on platelet production and related mechanisms unclear. Estrogen is a steroid hormone, mainly through estrogen receptor alpha and beta ER play ER effect of.ER alpha and ER beta is a nuclear receptor, not activated, ER is located on the cell membrane, activation of estrogen binding ligands, and two homodimers (ER alpha /ER alpha or beta ER /ER beta two) or heterologous dimer (ER alpha /ER beta), then shift from the cell membrane to in the nucleus, and the target gene promoter upstream of the estrogen response element (estrogen response sub element, ERE) with the expression and regulation of target genes, which play the biological effects of.ER alpha and ER beta distribution of.ER alpha as distinct tissue specificity in systemic manifestations mainly expressed in The sex organs such as ovarian, breast, and beta ER was mainly expressed in non sexual organs such as bone marrow, lung, colon and so on. In addition, ER alpha and ER beta in the structure and function as ligand recognition and binding, receptor activation, coactivator or corepressor recruitment target gene transcription regulation is also obvious the difference. It has been reported that ER alpha and ER beta were expressed in megakaryocytes but target molecules and signal transduction pathways which play a major role in the downstream receptor and estrogen on megakaryocyte function remain to be confirmed. On the other hand, estrogen is a classic anti radiation drug, in animal experiments have been confirmed with radiation protection, according to whether administered before or after exposure to drugs can significantly improve the survival rate. The same given estrogen in patients undergoing radiotherapy before irradiation, can reduce the peripheral white blood cells decreased, but estrogen can be used for the prevention and treatment of radiation Injury of thrombocytopenia still lack of relevant research. Based on the above scientific issues, this study used mouse bone marrow (bone marrow BM) primary megakaryocyte Sca1+ cells derived from human umbilical cord blood (cord, blood, CB) primary megakaryocytes derived from CD34+ cells, cells of human megakaryocyte progenitor cell line M07e. Mature megakaryocyte cell line Meg-01 and c-Mpl-/- and ER beta - knockout mice, using normal C57BL/6J mice of sublethal radiation damage reduced platelet amount in model, and give the estrogen treatment before irradiation and after irradiation, to observe the effect of estrogen on the recovery of platelet levels; the cell viability analysis, flow cytometry for immunofluorescence, analysis of estrogen on proliferation and differentiation of megakaryocytes, polyploid formation of laser confocal microscopy and blood testing technology, the influence of platelet precursor formation and platelet, to Clear regulatory effect of estrogen on platelet production and action stage; finally, using Western blot, RT-PCR Q, RT-PCR, small interfering RNA (Si RNA) gene knockdown, luciferase reporter assay, chromatin immunoprecipitation (Ch IP) and other methods, in-depth analysis of the molecular mechanism of promoting platelet formation of estrogen the main research results and conclusions are as follows: 1, through the establishment of acute radiation injury in mice found that acute radiation injury may lead to platelet levels in mice decreased rapidly, while estrogen pretreatment before radiation or radiation after estrogen intervention can effectively accelerate the recovery of platelet levels in mice after radiation, shorten the duration of platelet trough radiation after, and significantly improve the survival rate of mice after irradiation. The traditional anti radiation drugs can not only prevent radiation injury of thrombocytopenia, thrombocytopenia with radiation injury The treatment effect of.2 to some extent, although estrogen can not promote megakaryocyte progenitor cell proliferation, but can promote the differentiation of megakaryocyte maturation, polyploid formation, the formation of the platelet and platelet, and can promote bone marrow megakaryocyte differentiation, significantly increased peripheral blood platelet levels, suggesting that estrogen can to promote megakaryocyte differentiation advanced, so as to promote the formation of.3 platelets, megakaryocytes and estrogen treatment can significantly activate ER receptor megakaryocytes, and non ERa receptors, blocking or knockdown of ER beta can significantly inhibit estrogen induced megakaryocytic differentiation and polyploid formation, suggesting that ER may be the main target receptor beta.4 estrogen thrombopoietic effect, megakaryocyte ER beta activation induced by estrogen, megakaryocyte in many important regulatory molecules found in GATA1 was significantly up-regulated, while knockdown of ER beta Almost completely inhibited the estrogen induced upregulation of GATA1, suggesting that GATA1 may be the main target of estrogen effect. Further downstream by dual luciferase reporter system and chromatin immunoprecipitation (Ch IP) confirmed that ER and GATA1 can be beta promoter binding, ERE sequence can be located in the -1380 in addition to -1574; that knockdown of megakaryocyte GATA1 after estrogen induced megakaryocytic polyploidization effect disappeared. These results suggest that GATA1 is a downstream target molecules of ER beta, ER beta activation after binding to GATA1 promoter, which induced the expression of GATA1 was up-regulated, thereby promoting megakaryocyte polyploidy.5 in vitro also, estrogen can induce GATA1 downstream target molecule upregulation of STAT1 expression and activation; knockdown of megakaryocyte ER beta and GATA1, and up regulate the expression of estrogen and activation induced by STAT1 not; in addition, knockdown of STA The expression of T1 can be almost completely inhibited by estrogen promote polyploid formation, suggesting that STAT1 may be the downstream target of GATA1, mediates the effect of estrogen on.6 megacaryocyte polyploid formation, NF-E2 is a downstream target molecules of GAT1, in vitro experiments show that estrogen treatment can upregulate the expression of megakaryocytes after induced by NF-E2 however, knockdown of GATA1 can completely counteract estrogen induced upregulation of NF-E2; in addition, NF-E2 is the megakaryocyte precursors to the formation of key regulatory molecules and platelet production, these results suggest that NF-E2 may be the downstream target of GATA1, mediated by estrogen enhances platelet precursor formation and platelet production. Through this study, we not only reveal the estrogen in radiation injury prevention and treatment effect of thrombocytopenia disease, and estrogen in platelet production in the The mechanism of estrogen promotes megakaryocyte polyploidy formation and platelet production, and provides new ideas and new methods for the prevention and treatment of thrombocytopenia.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R818

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