本文選題：巨核細(xì)胞　切入點(diǎn)：生長(zhǎng)激素　出處：《第三軍醫(yī)大學(xué)》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：隨著電離輻射在軍事、經(jīng)濟(jì)、農(nóng)業(yè)生產(chǎn)、醫(yī)療衛(wèi)生等領(lǐng)域中的不斷應(yīng)用，由電離輻射引起的各種損傷也在逐漸增加。骨髓是輻射敏感組織之一，當(dāng)機(jī)體受到全身或局部一定劑量射線(xiàn)照射后，即可引起骨髓造血功能障礙，，表現(xiàn)為白細(xì)胞、紅細(xì)胞、血小板等全血細(xì)胞急劇減少，其中血小板生成減少和功能障礙是引起機(jī)體出血、感染的重要原因，也是影響機(jī)體存活和疾病預(yù)后的主要因素之一[1]。因此，促進(jìn)外周血血小板水平的快速恢復(fù)是治療輻射損傷的一個(gè)關(guān)鍵環(huán)節(jié)。目前臨床上治療血小板減少癥的藥物種類(lèi)較少，而且大部分還存在著起效慢、副作用明顯等缺點(diǎn)。所以，尋找和研制高效、低毒的新型升血小板藥物成為一個(gè)人們迫切關(guān)注的問(wèn)題。 血小板來(lái)源于骨髓巨核細(xì)胞，而巨核細(xì)胞又是經(jīng)造血干細(xì)胞分化而成，因此，巨核細(xì)胞的分化過(guò)程會(huì)直接影響血小板的產(chǎn)生時(shí)間和產(chǎn)量[2]。血小板生成素(thrombopoietin, TPO)是調(diào)節(jié)巨核細(xì)胞增殖、分化和血小板生成最重要的細(xì)胞因子。TPO與其受體c-Mpl結(jié)合后，激活相應(yīng)信號(hào)通路，從而刺激巨核細(xì)胞的增殖、分化和血小板生成[3]。但是有文獻(xiàn)報(bào)道，一些重度血小板減少癥患者在使用重組人TPO（rhTPO）后5天左右時(shí)間外周血血小板水平才開(kāi)始升高，到12天達(dá)到峰值，正因如此，患者處于血小板危象期的時(shí)間并沒(méi)有得到顯著縮短[4][5]。導(dǎo)致血小板延遲升高的原因可能與TPO不能促進(jìn)巨核細(xì)胞終末分化（包括血小板前體形成和血小板釋放）有關(guān)。更重要的是，由于能夠誘導(dǎo)體內(nèi)產(chǎn)生TPO中和性抗體[6][7]，美國(guó)FDA禁止了有關(guān)重組TPO基因工程藥物的研發(fā)�；诖�，人們轉(zhuǎn)而尋找和研制TPO的類(lèi)似物和模擬物。TPO模擬肽（thrombopoietin mimetic peptide, TMP）的發(fā)現(xiàn)引起了人們的充分關(guān)注[1]，雖然它僅含14個(gè)氨基酸，但其二聚體或二聯(lián)體多肽與TPO一樣都可以結(jié)合并激活TPO的受體c-Mpl，顯示出較強(qiáng)的促巨核細(xì)胞增殖與分化活性[2][3]。但是，無(wú)論是TMP二聚體還是二聯(lián)體多肽的分子量都比較小，體內(nèi)半衰期相對(duì)較短，使其體內(nèi)應(yīng)用受到限制。 研究證實(shí)，生長(zhǎng)激素(growth hormone, GH)通過(guò)與其受體GHR結(jié)合，能夠促進(jìn)不同種類(lèi)細(xì)胞的增殖和/或分化[4]。有研究發(fā)現(xiàn)體內(nèi)缺乏GH的DW/J侏儒小鼠外周血數(shù)量（包括有白細(xì)胞，紅細(xì)胞以及血小板）顯著降低[5]。隨后有報(bào)道指出，給予放/化療或骨髓移植小鼠大劑量的GH能夠顯著促進(jìn)小鼠骨髓造血功能重建，包括血小板水平的快速恢復(fù)[6][7][8][9][10]。另外，近來(lái)一項(xiàng)臨床研究結(jié)果表明，接受化療藥物強(qiáng)化治療的血液腫瘤患者在使用大劑量GH后其外周血小板水平能提前3天左右時(shí)間恢復(fù)[11]。所有的這些數(shù)據(jù)都提示GH可能在血小板生成過(guò)程中發(fā)揮了重要的調(diào)控作用，但其具體作用于血小板生成的哪個(gè)階段及相關(guān)機(jī)制尚不清楚。 本研究首先分析了GH和一種血小板生成素模擬肽二聯(lián)體(dTMP)分別對(duì)巨核細(xì)胞增殖分化、血小板前體形成以及血小板產(chǎn)生的調(diào)控作用及可能機(jī)制。研究發(fā)現(xiàn)dTMP主要作用于巨核細(xì)胞增殖分化的早期階段，而GH則主要作用于巨核細(xì)胞的終末分化階段，二者聯(lián)合具有協(xié)調(diào)促血小板生成的作用。在此基礎(chǔ)上，將GH與dTMP進(jìn)行融合，通過(guò)體外實(shí)驗(yàn)對(duì)dTMP-GH融合蛋白的促血小板生成作用進(jìn)行驗(yàn)證，進(jìn)而探討了dTMP-GH融合蛋白對(duì)輻射損傷所致小鼠血小板減少的救治作用。所取得的主要研究結(jié)果與結(jié)論如下： 1.采用免疫磁珠分離、流式細(xì)胞分析、免疫熒光、電鏡觀察、Western blot和CCK-8等方法，建立了人臍血來(lái)源巨核細(xì)胞原代培養(yǎng)、巨核細(xì)胞增殖分化、血小板前體形成和血小板產(chǎn)生等一套完整的血小板生成檢測(cè)與分析技術(shù)體系。 2.體外研究結(jié)果表明，GH不能促進(jìn)巨核祖細(xì)胞的增殖，但具有促進(jìn)巨核細(xì)胞分化的作用。 3. GH能夠以劑量依賴(lài)方式促進(jìn)晚期巨核細(xì)胞形成血小板前體和產(chǎn)生血小板，提示GH具有促進(jìn)巨核細(xì)胞終末分化的作用。 4.對(duì)于早期巨核祖細(xì)胞，GH不能激活STAT5信號(hào)通路，但能延遲和持續(xù)激活ERK1/2信號(hào)通路，給予ERK1/2信號(hào)通路阻斷劑能夠抑制GH的促巨核細(xì)胞分化作用，提示GH的促巨核細(xì)胞分化作用與其激活ERK1/2的方式有關(guān)。 5.對(duì)于晚期成熟巨核細(xì)胞，GH具有快速激活A(yù)kt信號(hào)通路的作用，并且能夠增強(qiáng)Rho激酶Cdc42和Rac1的活性，給予Akt和Rho信號(hào)通路阻斷劑可以抑制GH的促血小板前體形成和血小板生成作用，提示GH通過(guò)激活A(yù)kt/Rho信號(hào)通路而發(fā)揮促進(jìn)巨核細(xì)胞終末分化的功能。 6.體外實(shí)驗(yàn)證實(shí)dTMP能夠以劑量依賴(lài)方式促進(jìn)巨核祖細(xì)胞的增殖與分化，但對(duì)血小板前體形成和產(chǎn)板卻具有抑制作用，說(shuō)明dTMP主要通過(guò)促進(jìn)早期巨核細(xì)胞的增殖與分化而發(fā)揮促血小板生成作用。 7.與單獨(dú)dTMP相比，dTMP與GH聯(lián)合應(yīng)用能顯著促進(jìn)血小板前體形成、加速血小板生成與釋放，同時(shí)伴有β1-tubulin表達(dá)增強(qiáng)、巨核細(xì)胞內(nèi)膜系統(tǒng)形成，提示GH與dTMP在血小板生成方面具有互補(bǔ)作用。 8. dTMP-GH融合蛋白可以顯著促進(jìn)巨核細(xì)胞增殖與分化，且其作用顯著優(yōu)于單純dTMP以及dTMP+GH處理組，提示通過(guò)與GH融合可以提高dTMP的促巨核細(xì)胞增殖分化能力，其機(jī)制可能與蛋白空間構(gòu)象改變或受體介導(dǎo)信號(hào)通路的交叉激活有關(guān)。 9. dTMP-GH融合蛋白能夠顯著上調(diào)巨核細(xì)胞GATA-1、NF-E2及β1-tubulin的表達(dá)；與單純dTMP處理組相比，dTMP-GH融合蛋白處理組血小板前體形成和血小板產(chǎn)生時(shí)間提前，數(shù)量增多。 10. Western blot結(jié)果表明，dTMP-GH不僅能夠快速激活巨核祖細(xì)胞中STAT5信號(hào)通路，而且還能持續(xù)活化ERK1/2信號(hào)通路，同時(shí)還具有激活晚期巨核細(xì)胞Akt的能力，從信號(hào)通路激活角度進(jìn)一步證實(shí)dTMP-GH同時(shí)具有促進(jìn)早期巨核細(xì)胞增殖分化和晚期巨核細(xì)胞成熟和產(chǎn)板的活性。 11.成功復(fù)制急性輻射損傷小鼠血小板減少癥動(dòng)物模型，與生理鹽水對(duì)照組和dTMP處理組相比，給予dTMP-GH融合蛋白處理能夠加速受照小鼠血小板水平恢復(fù)，顯著升高受照小鼠外周血小板最低值水平，縮短血小板處于低谷期時(shí)間，并顯著提高小鼠存活率，提示dTMP-GH對(duì)急性輻射損傷所致血小板減少具有突出的救治作用。 12. dTMP-GH融合蛋白對(duì)卡鉑化療及放療聯(lián)合化療所致小鼠血小板減少也具有顯著的救治作用，不僅可以促進(jìn)血小板水平快速恢復(fù)，而且還可顯著降低小鼠死亡率。 總之，通過(guò)本實(shí)驗(yàn)研究我們首次發(fā)現(xiàn)GH具有促進(jìn)巨核細(xì)胞終末分化、加速血小板前體形成和血小板產(chǎn)生的作用，并初步闡明了其作用機(jī)理；同時(shí)，發(fā)現(xiàn)GH與dTMP具有協(xié)同促血小板生成的作用，并證實(shí)dTMP-GH重組融合蛋白同時(shí)具有促進(jìn)早期巨核細(xì)胞增殖、分化和晚期巨核細(xì)胞成熟和終末分化的活性，對(duì)輻射損傷和化療所致血小板減少具有突出的救治作用，值得進(jìn)一步研發(fā)。
[Abstract]:With ionizing radiation in the military, economic, agricultural production, continuous application in medical and health fields, various kinds of damage caused by ionizing radiation is also gradually increased. The bone marrow is one of the radiation sensitive tissue, when the body by systemic or local irradiation dose radiation, can cause bone marrow hematopoietic dysfunction, manifested as white blood cells, red blood cells and platelet blood cells decreased rapidly, which decreased platelet production and dysfunction is caused by bleeding, an important cause of infection, it is also a main factor affecting survival and disease prognosis of [1]. therefore, promote the rapid recovery of the level of platelet in peripheral blood is a key to the treatment of radiation injury. The clinical medicine for treating thrombocytopenic fewer types of disease, and most are slow onset, obvious side effects and other shortcomings. Therefore, to find and develop new high efficiency and low toxicity. Platelets have become an urgent concern.
Platelet derived from bone marrow megakaryocytes and megakaryocytes and the cell differentiation of hematopoietic stem and therefore, differentiation of megakaryocytes will directly affect platelet production time and the production of thrombopoietin (thrombopoietin, TPO) [2]. is the regulation of megakaryocyte proliferation, differentiation and platelet production of the most important cytokines and.TPO c-Mpl receptor binding, activate the corresponding signaling pathway, thereby stimulating megakaryocyte proliferation, differentiation and platelet production but [3]. reported in the literature, some severe thrombocytopenia in patients using recombinant human TPO (rhTPO) level of platelet in peripheral blood after 5 days of time began to increase, reached the peak on the 12 day, because so, in patients with platelet crisis period has not been significantly shorten the delay increase reasons may lead to platelet and megakaryocyte TPO cannot promote cell terminal differentiation ([4][5]. package The platelet precursor formation and platelet release). What is more important, because it can produce TPO neutralizing antibodies in vivo induced by [6][7], the FDA banned the R & D of recombinant TPO gene engineering medicine. Based on this, we turn to find and develop TPO analogs and mimetics of.TPO mimetic peptide (thrombopoietin mimetic peptide, TMP) that causes people's attention [1], although it only contains 14 amino acids, but its dimer or two cis polypeptide like TPO can bind and activate the TPO receptor c-Mpl, showed proliferation and differentiation activity of [2][3]. megakaryocyte strong but both dimer molecule TMP two or two the amount of cis peptide are relatively small, the body has a relatively short half-life in vivo, its application is limited.
Study confirmed that growth hormone (growth hormone, GH) via binding with GHR can promote different types of cell proliferation and / or differentiation of [4]. research has found that the lack of GH DW/J dwarf mice (including the number of peripheral blood leukocytes, red blood cells and platelets) [5]. then reported significantly lower, to give / chemotherapy or bone marrow transplantation in mice of high dose GH could significantly promote hematopoietic reconstruction, rapid recovery of [6][7][8][9][10]. also includes platelet levels, a recent clinical study showed that chemotherapy drugs strengthen blood of cancer patients on their use of high dose GH weeks after platelet levels 3 days in advance all the time to recover [11]. these data suggest that GH may play an important role in the regulation of platelet production process, but its specific role in which platelet formation The stages and related mechanisms are not yet clear.
This paper first analyzes the GH and a thrombopoietin simulated peptide two CIS (dTMP) respectively on the proliferation of megakaryocytic differentiation, regulation of platelet precursor formation and platelet production and possible mechanisms. The study found that the main function of dTMP at early stages of megakaryocyte proliferation and differentiation, while GH is a major role in the terminal differentiation stage megakaryocytes, two combined with the coordination of thrombopoietic effect. Based on this, GH and dTMP were fused by in vitro experiments to verify the thrombopoietic effect of dTMP-GH fusion protein, and discusses the effects of dTMP-GH fusion protein in mice induced by treatment of thrombocytopenia on radiation injury. The main results the conclusions are as follows:
1. using immunomagnetic separation, flow cytometry analysis, immunofluorescence, electron microscopy, Western blot and CCK-8, was established from human umbilical cord blood megakaryocyte cell primary culture, proliferation and differentiation of megakaryocytes and platelet precursor formation and platelet platelet a complete build detection and analysis system.
2. the results of in vitro study showed that GH could not promote the proliferation of megakaryocyte progenitor cells, but could promote the differentiation of megakaryocyte.
3. GH can promote the formation of platelets and platelet in advanced megakaryocytes in a dose dependent manner, suggesting that GH can promote the terminal differentiation of megakaryocytes.
4. for early megakaryocyte progenitor cells, GH can activate the STAT5 signaling pathway, but delayed and sustained activation of ERK1/2 signal pathway, ERK1/2 signal pathway blocker can promote megakaryocyte differentiation inhibition of GH, suggesting GH megacaryocyte differentiation and activation of the ERK1/2 mode.
5. for late mature megakaryocytes, GH has a rapid activation of Akt signaling pathway and Rho kinase can enhance Cdc42 and Rac1 activity, given Akt and Rho signal pathway blocker can promote platelet platelet precursor formation and inhibit the generation of GH, suggesting that GH through activation of Akt/Rho signaling pathway and promote terminal megakaryocyte function.
6. in vitro, it is confirmed that dTMP can promote proliferation and differentiation of megakaryocyte progenitor cells in a dose dependent manner, but inhibit the formation of platelets precursors and production plates, indicating that dTMP plays a role in promoting platelet production by promoting the proliferation and differentiation of megakaryocytes.
7., compared with dTMP alone, combined application of dTMP and GH can significantly promote platelet precursor formation, accelerate platelet production and release, accompanied by enhanced expression of beta 1-tubulin and formation of megakaryocyte inner membrane system, suggesting that GH and dTMP have complementary roles in platelet production.
8. dTMP-GH fusion protein can significantly promote the proliferation and differentiation of megakaryocytes, and its effect was better than that of pure dTMP and dTMP+GH treatment group, indicating that the fusion of GH and can improve the ability to promote the proliferation and differentiation of megakaryocytes of dTMP, its mechanism may be related to protein conformational changes or receptor mediated pathway cross activation.
9. dTMP-GH fusion protein significantly up-regulated the expression of GATA-1, NF-E2 and beta 1-tubulin in megakaryocytes. Compared with the dTMP treatment group, the platelet precursor formation and platelet production time in the dTMP-GH fusion protein treatment group increased earlier.
10. Western blot results show that dTMP-GH can not only activate the STAT5 signaling pathway of megakaryocyte progenitor cells, but also the persistent activation of ERK1/2 signaling pathway, but also has the ability to activate late megakaryocyte Akt, further confirmed that dTMP-GH can promote the proliferation and differentiation of megakaryocytes early and late megakaryocyte maturation and the production activity from the board the activation of signaling pathway analysis.
11. the success of acute radiation injury in mice with thrombocytopenia animal model, and compared with the saline control group and dTMP treatment group, given treatment with dTMP-GH fusion protein can accelerate the exposure level of mice significantly increased platelet recovery by platelet in peripheral according to minimum level, shorten the time of platelet in the trough, and significantly improve the survival rate of mice. DTMP-GH on acute radiation injury has prominent therapeutic effect caused by reduced platelet.
12. dTMP-GH fusion protein also has significant therapeutic effect on thrombocytopenia induced by carboplatin chemotherapy and radiotherapy combined with chemotherapy. It can not only promote the rapid recovery of platelet level, but also significantly reduce the mortality of mice.
In short, through this study we first found that GH can promote the terminal differentiation of megakaryocytes, accelerate the platelet precursor formation and platelet effect, and clarify its mechanism of action; at the same time, found that GH and dTMP have synergistic thrombopoietic effect, and confirmed that dTMP-GH recombinant fusion protein can promote the proliferation and early megakaryocyte, activity of megakaryocyte maturation and terminal differentiation and differentiation stage, reduce the effect of treatment with serious radiation damage and platelet induced by chemotherapy, and is worthy of further research.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R818

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