GH促血小板生成机制分析及dTMP-GH对辐射损伤小鼠的救治作用研究

发布时间：2018-03-19 14:06

本文选题：巨核细胞　切入点：生长激素　出处：《第三军医大学》2014年博士论文　论文类型：学位论文

【摘要】：随着电离辐射在军事、经济、农业生产、医疗卫生等领域中的不断应用，由电离辐射引起的各种损伤也在逐渐增加。骨髓是辐射敏感组织之一，当机体受到全身或局部一定剂量射线照射后，即可引起骨髓造血功能障碍，，表现为白细胞、红细胞、血小板等全血细胞急剧减少，其中血小板生成减少和功能障碍是引起机体出血、感染的重要原因，也是影响机体存活和疾病预后的主要因素之一[1]。因此，促进外周血血小板水平的快速恢复是治疗辐射损伤的一个关键环节。目前临床上治疗血小板减少症的药物种类较少，而且大部分还存在着起效慢、副作用明显等缺点。所以，寻找和研制高效、低毒的新型升血小板药物成为一个人们迫切关注的问题。血小板来源于骨髓巨核细胞，而巨核细胞又是经造血干细胞分化而成，因此，巨核细胞的分化过程会直接影响血小板的产生时间和产量[2]。血小板生成素(thrombopoietin, TPO)是调节巨核细胞增殖、分化和血小板生成最重要的细胞因子。TPO与其受体c-Mpl结合后，激活相应信号通路，从而刺激巨核细胞的增殖、分化和血小板生成[3]。但是有文献报道，一些重度血小板减少症患者在使用重组人TPO（rhTPO）后5天左右时间外周血血小板水平才开始升高，到12天达到峰值，正因如此，患者处于血小板危象期的时间并没有得到显著缩短[4][5]。导致血小板延迟升高的原因可能与TPO不能促进巨核细胞终末分化（包括血小板前体形成和血小板释放）有关。更重要的是，由于能够诱导体内产生TPO中和性抗体[6][7]，美国FDA禁止了有关重组TPO基因工程药物的研发。基于此，人们转而寻找和研制TPO的类似物和模拟物。TPO模拟肽（thrombopoietin mimetic peptide, TMP）的发现引起了人们的充分关注[1]，虽然它仅含14个氨基酸，但其二聚体或二联体多肽与TPO一样都可以结合并激活TPO的受体c-Mpl，显示出较强的促巨核细胞增殖与分化活性[2][3]。但是，无论是TMP二聚体还是二联体多肽的分子量都比较小，体内半衰期相对较短，使其体内应用受到限制。研究证实，生长激素(growth hormone, GH)通过与其受体GHR结合，能够促进不同种类细胞的增殖和/或分化[4]。有研究发现体内缺乏GH的DW/J侏儒小鼠外周血数量（包括有白细胞，红细胞以及血小板）显著降低[5]。随后有报道指出，给予放/化疗或骨髓移植小鼠大剂量的GH能够显著促进小鼠骨髓造血功能重建，包括血小板水平的快速恢复[6][7][8][9][10]。另外，近来一项临床研究结果表明，接受化疗药物强化治疗的血液肿瘤患者在使用大剂量GH后其外周血小板水平能提前3天左右时间恢复[11]。所有的这些数据都提示GH可能在血小板生成过程中发挥了重要的调控作用，但其具体作用于血小板生成的哪个阶段及相关机制尚不清楚。本研究首先分析了GH和一种血小板生成素模拟肽二联体(dTMP)分别对巨核细胞增殖分化、血小板前体形成以及血小板产生的调控作用及可能机制。研究发现dTMP主要作用于巨核细胞增殖分化的早期阶段，而GH则主要作用于巨核细胞的终末分化阶段，二者联合具有协调促血小板生成的作用。在此基础上，将GH与dTMP进行融合，通过体外实验对dTMP-GH融合蛋白的促血小板生成作用进行验证，进而探讨了dTMP-GH融合蛋白对辐射损伤所致小鼠血小板减少的救治作用。所取得的主要研究结果与结论如下： 1.采用免疫磁珠分离、流式细胞分析、免疫荧光、电镜观察、Western blot和CCK-8等方法，建立了人脐血来源巨核细胞原代培养、巨核细胞增殖分化、血小板前体形成和血小板产生等一套完整的血小板生成检测与分析技术体系。 2.体外研究结果表明，GH不能促进巨核祖细胞的增殖，但具有促进巨核细胞分化的作用。 3. GH能够以剂量依赖方式促进晚期巨核细胞形成血小板前体和产生血小板，提示GH具有促进巨核细胞终末分化的作用。 4.对于早期巨核祖细胞，GH不能激活STAT5信号通路，但能延迟和持续激活ERK1/2信号通路，给予ERK1/2信号通路阻断剂能够抑制GH的促巨核细胞分化作用，提示GH的促巨核细胞分化作用与其激活ERK1/2的方式有关。 5.对于晚期成熟巨核细胞，GH具有快速激活Akt信号通路的作用，并且能够增强Rho激酶Cdc42和Rac1的活性，给予Akt和Rho信号通路阻断剂可以抑制GH的促血小板前体形成和血小板生成作用，提示GH通过激活Akt/Rho信号通路而发挥促进巨核细胞终末分化的功能。 6.体外实验证实dTMP能够以剂量依赖方式促进巨核祖细胞的增殖与分化，但对血小板前体形成和产板却具有抑制作用，说明dTMP主要通过促进早期巨核细胞的增殖与分化而发挥促血小板生成作用。 7.与单独dTMP相比，dTMP与GH联合应用能显著促进血小板前体形成、加速血小板生成与释放，同时伴有β1-tubulin表达增强、巨核细胞内膜系统形成，提示GH与dTMP在血小板生成方面具有互补作用。 8. dTMP-GH融合蛋白可以显著促进巨核细胞增殖与分化，且其作用显著优于单纯dTMP以及dTMP+GH处理组，提示通过与GH融合可以提高dTMP的促巨核细胞增殖分化能力，其机制可能与蛋白空间构象改变或受体介导信号通路的交叉激活有关。 9. dTMP-GH融合蛋白能够显著上调巨核细胞GATA-1、NF-E2及β1-tubulin的表达；与单纯dTMP处理组相比，dTMP-GH融合蛋白处理组血小板前体形成和血小板产生时间提前，数量增多。 10. Western blot结果表明，dTMP-GH不仅能够快速激活巨核祖细胞中STAT5信号通路，而且还能持续活化ERK1/2信号通路，同时还具有激活晚期巨核细胞Akt的能力，从信号通路激活角度进一步证实dTMP-GH同时具有促进早期巨核细胞增殖分化和晚期巨核细胞成熟和产板的活性。 11.成功复制急性辐射损伤小鼠血小板减少症动物模型，与生理盐水对照组和dTMP处理组相比，给予dTMP-GH融合蛋白处理能够加速受照小鼠血小板水平恢复，显著升高受照小鼠外周血小板最低值水平，缩短血小板处于低谷期时间，并显著提高小鼠存活率，提示dTMP-GH对急性辐射损伤所致血小板减少具有突出的救治作用。 12. dTMP-GH融合蛋白对卡铂化疗及放疗联合化疗所致小鼠血小板减少也具有显著的救治作用，不仅可以促进血小板水平快速恢复，而且还可显著降低小鼠死亡率。总之，通过本实验研究我们首次发现GH具有促进巨核细胞终末分化、加速血小板前体形成和血小板产生的作用，并初步阐明了其作用机理；同时，发现GH与dTMP具有协同促血小板生成的作用，并证实dTMP-GH重组融合蛋白同时具有促进早期巨核细胞增殖、分化和晚期巨核细胞成熟和终末分化的活性，对辐射损伤和化疗所致血小板减少具有突出的救治作用，值得进一步研发。
[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.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R818

【参考文献】