本文關(guān)鍵詞： 雌激素 電離輻射 血小板減少癥 巨核細(xì)胞 多倍體形成 GATA1 血小板生成　出處：《第三軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：隨著科技的發(fā)展,核能及其相關(guān)的技術(shù)在軍事、醫(yī)學(xué)、工業(yè)等多個(gè)領(lǐng)域得到了更加廣泛的應(yīng)用。然而,核能為我們的生活帶來便利的同時(shí),也增加了由電離輻射引起放射損傷的潛在風(fēng)險(xiǎn)。尤其是核事故、核爆炸等情況下會產(chǎn)生大量的放射損傷傷員。此外,臨床上接受放射治療和骨髓移植的腫瘤患者也會受到一定程度的放射損傷。骨髓是放射損傷的主要靶器官。其中,放射損傷引起的血小板嚴(yán)重減少會導(dǎo)致機(jī)體出血和感染,甚至死亡。不幸的是,目前臨床上血小板減少癥的治療藥物種類較少,而且它們大多還存在著起效慢、副作用大、價(jià)格昂貴等缺點(diǎn)。因此,尋找一種有效、安全的放射損傷血小板減少癥的預(yù)防和救治方法顯得至關(guān)重要。血小板由骨髓中的巨核細(xì)胞產(chǎn)生,血小板生成過程主要包括造血干細(xì)胞定向分化為巨核祖細(xì)胞、巨核祖細(xì)胞大量擴(kuò)增并分化為成熟巨核細(xì)胞、成熟巨核細(xì)胞釋放血小板等多個(gè)階段。其中巨核細(xì)胞的分化成熟是血小板產(chǎn)生的關(guān)鍵步驟。巨核細(xì)胞成熟首先進(jìn)行核內(nèi)有絲分裂以形成多倍體,然后巨核細(xì)胞進(jìn)入一個(gè)胞質(zhì)成熟過程,即脂質(zhì)和蛋白質(zhì)大量合成并形成分界膜系統(tǒng),最后巨核細(xì)胞向血管竇伸出血小板前體并向血液循環(huán)中釋放血小板。在體內(nèi),調(diào)控血小板生成最重要的細(xì)胞因子是血小板生成素(Thrombopoietin,TPO)。但是,我們及他人的研究發(fā)現(xiàn)體內(nèi)還有其他因子也參與了血小板生成的調(diào)控。雌激素是哺乳動物體內(nèi)非常重要的一種性激素,主要由卵巢產(chǎn)生和分泌,它在人體內(nèi)有著廣泛的生物學(xué)作用。多個(gè)研究報(bào)道,雌激素對造血系統(tǒng)也有影響作用。有趣的是,巨核細(xì)胞不但被證實(shí)自身能產(chǎn)生雌激素,而且雌激素和巨核細(xì)胞生成和血小板生成有一定的聯(lián)系。大量的臨床數(shù)據(jù)表明人體血小板水平也存在性別差異,而且這種性別差異與體內(nèi)的雌激素水平有關(guān)。另外,大量的研究表明,雌激素不僅在體外能夠促進(jìn)巨核細(xì)胞分化及血小板前體形成,在體內(nèi)也可促進(jìn)血小板的產(chǎn)生,但是使用他莫昔芬或氟維司群抑制雌激素受體(estrogen receptor,ER)的激活則會抑制血小板的產(chǎn)生。這些研究表明雌激素參與了巨核細(xì)胞的生成和血小板的產(chǎn)生,但是雌激素作用于血小板生成的哪一階段及其相關(guān)機(jī)制仍未完全闡明。雌激素是一種類固醇激素,主要通過雌激素受體ERα和ERβ發(fā)揮作用。ERα和ERβ是一類核受體,未激活時(shí),ER位于細(xì)胞膜上,與配體雌激素結(jié)合后激活,從而形成同源二聚體(ERα/ERα或ERβ/ERβ)或異源二聚體(ERα/ERβ),然后由細(xì)胞膜移位到細(xì)胞核中,與靶基因啟動子上游的雌激素反應(yīng)元件(estrogen response element,ERE)結(jié)合而調(diào)節(jié)靶基因的表達(dá),從而發(fā)揮其生物學(xué)效應(yīng)。ERα和ERβ在全身表現(xiàn)為明顯的組織特異性分布。ERα主要表達(dá)于性器官如卵巢、乳腺等,而ERβ主要表達(dá)于非性腺器官如骨髓、肺、結(jié)腸等。另外,ERα和ERβ在結(jié)構(gòu)及功能如配體識別和結(jié)合、受體激活、輔活化子或輔阻遏物的募集、靶基因轉(zhuǎn)錄調(diào)節(jié)上也有明顯的差異。已有研究報(bào)道,巨核細(xì)胞均表達(dá)ERα和ERβ,但是,哪種受體在雌激素對巨核細(xì)胞的作用中發(fā)揮主要作用及其下游的靶分子和信號轉(zhuǎn)導(dǎo)通路仍有待證實(shí)。另一方面,雌激素是一種經(jīng)典的抗放藥物,在動物實(shí)驗(yàn)中都被證實(shí)有輻射保護(hù)作用,無論照前給藥還是照后給藥都可以顯著提高存活率。同樣在放療病人照射前給予雌激素,可以減輕外周血白細(xì)胞下降的程度,但是雌激素能否用于預(yù)防和治療放射損傷血小板減少癥仍然缺乏相關(guān)的研究。圍繞以上科學(xué)問題,本研究使用小鼠骨髓(bone marrow,BM)Sca1+細(xì)胞來源的原代巨核細(xì)胞、人臍帶血(cord blood,CB)CD34+細(xì)胞來源的原代巨核細(xì)胞、人巨核祖細(xì)胞株M07e細(xì)胞、人成熟巨核細(xì)胞株Meg-01細(xì)胞及c-Mpl-/-和ERβ-/-敲除小鼠,首先利用正常的C57BL/6J小鼠建立亞致死量放射損傷血小板減少癥模型,并在照射前或照射后給予雌激素處理,觀察雌激素對血小板水平恢復(fù)的影響;再利用細(xì)胞活性分析、流式細(xì)胞術(shù)、細(xì)胞免疫熒光、激光共聚焦顯微鏡觀察和血常規(guī)檢測等技術(shù)分析雌激素對巨核細(xì)胞增殖、分化、多倍體形成、血小板前體形成及血小板產(chǎn)生的影響,以明確雌激素對血小板生成的調(diào)控作用及作用階段;最后,再利用western blot、RT-PCR、q RT-PCR、小干擾RNA(si RNA)基因敲低、雙熒光素酶報(bào)告系統(tǒng)、染色質(zhì)免疫共沉淀(Ch IP)等方法,對雌激素促血小板生成作用的分子機(jī)制進(jìn)行深入分析。主要研究結(jié)果和結(jié)論如下:1、通過建立小鼠急性放射損傷模型發(fā)現(xiàn),急性放射損傷可導(dǎo)致小鼠血小板水平快速降低,而放射前雌激素預(yù)處理或放射后雌激素干預(yù)均能有效加速放射后小鼠血小板水平的恢復(fù),縮短放射后血小板低谷期的持續(xù)時(shí)間,并顯著提高受照后小鼠的生存率。說明傳統(tǒng)抗輻射藥物不但可以預(yù)防放射損傷血小板減少癥,還對放射損傷血小板減少癥有一定的救治作用。2、雌激素盡管不能促進(jìn)巨核祖細(xì)胞的增殖,但卻可以促進(jìn)巨核細(xì)胞的分化成熟、多倍體形成、血小板前體的形成和血小板的產(chǎn)生,并且可以促進(jìn)小鼠骨髓巨核細(xì)胞成熟分化,顯著升高外周血血小板水平,說明雌激素可以促進(jìn)巨核細(xì)胞晚期分化,從而促進(jìn)血小板的生成。3、雌激素處理巨核細(xì)胞可以顯著激活巨核細(xì)胞的ERβ受體,而非ERa受體,阻斷或敲低ERβ可以顯著抑制雌激素誘導(dǎo)的巨核細(xì)胞分化成熟和多倍體形成,提示ERβ可能是雌激素促血小板生成作用的主要靶受體。4、雌激素誘導(dǎo)巨核細(xì)胞ERβ激活后,在眾多巨核細(xì)胞重要調(diào)控分子中發(fā)現(xiàn)GATA1的表達(dá)顯著上調(diào),而敲低ERβ幾乎完全抑制了雌激素誘導(dǎo)的GATA1表達(dá)上調(diào),提示GATA1可能是雌激素下游效應(yīng)的主要靶分子。進(jìn)一步通過雙螢光素酶報(bào)告系統(tǒng)和染色質(zhì)免疫共沉淀(Ch IP)實(shí)驗(yàn)證實(shí)ERβ可以和GATA1的啟動子結(jié)合,可能的ERE序列位于-1380至-1574;另外,敲低巨核細(xì)胞GATA1的表達(dá)后,雌激素誘導(dǎo)巨核細(xì)胞多倍體形成效應(yīng)消失。這些結(jié)果表明,GATA1是ERβ下游的靶分子,ERβ激活后可與GATA1啟動子結(jié)合,從而誘導(dǎo)GATA1表達(dá)上調(diào),進(jìn)而促進(jìn)巨核細(xì)胞多倍體形成。5、體外實(shí)驗(yàn)證明,雌激素還可誘導(dǎo)GATA1下游靶分子STAT1表達(dá)上調(diào)和活化增強(qiáng);敲低巨核細(xì)胞ERβ和GATA1的表達(dá)后,雌激素并不能誘導(dǎo)STAT1表達(dá)上調(diào)和活化增強(qiáng);另外,敲低STAT1的表達(dá)幾乎可以完全抑制雌激素促多倍體形成的作用,提示STAT1可能是GATA1作用的下游靶分子,介導(dǎo)了雌激素促巨核細(xì)胞多倍體形成的作用。6、NF-E2是GAT1下游的一個(gè)靶分子,體外實(shí)驗(yàn)表明雌激素處理巨核細(xì)胞后可以誘導(dǎo)NF-E2的表達(dá)上調(diào),而敲低GATA1則可完全抵消雌激素誘導(dǎo)的NF-E2表達(dá)上調(diào);另外,NF-E2是巨核細(xì)胞血小板前體形成和血小板產(chǎn)生的關(guān)鍵調(diào)控分子,這些結(jié)果提示,NF-E2可能是GATA1作用的下游靶分子,介導(dǎo)了雌激素促血小板前體形成和血小板產(chǎn)生的作用。通過本實(shí)驗(yàn)研究,我們不僅揭示了雌激素在放射損傷血小板減少癥中的預(yù)防和救治作用,而且明確了雌激素在血小板生成中的作用,深入闡明了雌激素促進(jìn)巨核細(xì)胞多倍體形成和血小板產(chǎn)生的分子機(jī)制,從而為臨床血小板減少癥的預(yù)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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R818

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