rhIGF-Ⅰ促进辐射损伤小鼠巨核系造血功能恢复作用及机制研究

发布时间：2018-01-23 05:59

本文关键词： 胰岛素样生长因子-Ⅰ 辐照损伤造血恢复巨核细胞增殖凋亡　出处：《第三军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：电离辐射损伤不仅见于核武器爆炸、核恐怖袭击，由自然灾害或人为因素引起的核事故、核泄漏等情况下也常有发生，如日本广岛、长崎的原子弹爆炸及前苏联的切尔诺贝利核电站事故中都有数以万计的人员受到电离辐射损伤。骨髓是辐射敏感器官，大剂量电离辐射可引起骨髓多系造血功能障碍，包括红系、粒系、巨核系等造血细胞减少，并由此造成外周血红细胞、白细胞及血小板水平显著降低。人或动物受到辐射损伤后会出现出血、感染和物质代谢紊乱等多种症状，其中由造血功能障碍引起的血小板和白细胞减少是导致出血、感染乃至死亡的主要原因。经过多年研究，人们对放射损伤所致骨髓造血功能障碍的发生机理已有深刻认识，除了电离辐射可以直接损伤骨髓有核细胞外，其它因素还可以造成造血生成细胞的进一步减少，如在早期研究单纯放射损伤、放烧复合伤时，我室的程天民教授曾观察到大鼠和犬的骨髓巨核细胞可以被中性粒细胞噬食的现象，并将其命名为“巨核细胞被噬”(megakaryocytophagia)。因此，放射损伤后骨髓巨核细胞数量和外周血血小板水平降低尤为显著，如何促进放射损伤后骨髓造血功能恢复尤其是有效提升巨核细胞和血小板水平，是辐射损伤救治的一个关键环节。胰岛素样生长因子（IGF）最初由Rinderkencth于1976年从人血清中分离得到，目前发现IGF有两种，分别为IGF-Ⅰ和IGF-Ⅱ。IGF-Ⅰ在基因结构上与胰岛素原具有高度同源性，并且因具有类似胰岛素样作用而得名。IGF-Ⅰ是生长调节素的一种，由70个氨基酸的单链多肽组成，分子量为7.5kD，分子中含有3个二硫键。IGF-Ⅰ是生长激素的重要效应分子，其生成也主要受生长激素调控，研究表明生长激素能够刺激周围细胞分泌IGF-Ⅰ。正常情况下，机体内的IGF-Ⅰ主要由肝脏细胞、肾脏细胞、脾脏细胞等细胞内以旁分泌或自分泌方式产生，大部分IGF-Ⅰ与胰岛素样生长因子结合蛋白（IGFBP）结合而存在于血液中。IGF-Ⅰ主要通过其受体IGF-ⅠR介导发挥广泛的生物学作用，是细胞增殖的重要调控因子。研究表明重组人IGF-Ⅰ （rhIGF-Ⅰ）不仅具有促细胞增殖作用，而且还能够发挥抗细胞凋亡的功能，如rhIGF-Ⅰ对电离辐射诱导的唾液腺细胞和肠上皮细胞凋亡具有显著抑制作用。 rhIGF-Ⅰ在促进骨髓造血生成方面也发挥着重要作用，文献报道，在体外rhIGF-Ⅰ不仅能促进脐带血来源的CD34~+造血干细胞增殖，并且还能促进骨髓红系、粒系、淋巴系等多系集落的生成。然而，rhIGF-Ⅰ对巨核系细胞的生成是否也具有调控作用，尚未见到相关研究与报道。此外，体内应用rhIGF-Ⅰ是否能有效发挥促骨髓造血生成作用，并具有抗辐射诱导骨髓细胞凋亡的功能，这些都还缺乏深入系统研究。另一方面，在临床上，由放、化疗及其它原因引起的造血功能障碍也较为常见，如何有效促进骨髓造血功能恢复一直是临床研究的一个热点。因此，深入研究rhIGF-Ⅰ在辐射损伤后造血功能恢复过程中的作用，尤其是明确其对巨核系造血生成的调控作用与机制，不仅有助于进一步解读IGF-Ⅰ的功能，也可为研制新型造血生成调控药物提供思路与依据。本文重点研究了rhIGF-Ⅰ对辐射损伤小鼠巨核系造血功能恢复的促进作用及分子机制。选择BALB/c小鼠为研究对象，首先通过血象动态检测和组织病理切片观察，分析辐射损伤小鼠给予rhIGF-Ⅰ处理后外周血血细胞和骨髓有核细胞计数变化，重点观察rhIGF-Ⅰ处理对骨髓巨核细胞和外周血血小板水平的影响作用，，明确rhIGF-Ⅰ是否具有促进辐射损伤小鼠巨核系造血功能恢复的作用；分离骨髓单核细胞和造血干细胞，分析rhIGF-Ⅰ处理后巨核细胞分化标志分子的表达变化，观察rhIGF-Ⅰ是否可以促进造血干细胞向巨核细胞的分化；进一步以体外培养的巨核细胞株为研究对象，研究rhIGF-Ⅰ处理对巨核细胞增殖和辐照诱导巨核细胞凋亡的影响，以及巨核细胞骨架actin和β1-tubulin分布与重排变化，揭示其在促进巨核系造血功能恢复中的作用与机制。结论： 1、BALB/c小鼠受到7.5Gy γ射线全身一次性照射后立即给予100μg/kg的rhIGF-Ⅰ皮下注射，与辐照对照组相比，rhIGF-Ⅰ处理组小鼠40天存活率提高近20%，提示rhIGF-Ⅰ具有明显的辐射保护作用。 2、外周血血细胞计数结果表明，接受5.0Gy γ射线照射的小鼠，在给予rhIGF-Ⅰ处理后的第10-17天，除了红细胞和白细胞数量显著增多外，rhIGF-Ⅰ处理组小鼠的外周血血小板水平也显著高于辐照对照组；骨髓组织病理检测发现，放射损伤后给予rhIGF-Ⅰ处理，包括巨核细胞在内的骨髓有核细胞数目显著增多；集落形成实验发现，rhIGF-Ⅰ处理组小鼠CFU-GM、BFU-E和CFU-GEMM集落数均显著增多。这些结果综合提示rhIGF-Ⅰ具有促进辐射损伤小鼠包括巨核系在内的骨髓多系造血功能恢复的作用。 3、放射损伤后给予rhIGF-Ⅰ处理，小鼠骨髓c-kit~+细胞显著增多，进一步分析发现，rhIGF-Ⅰ可促进Lin~-c-kit~+细胞中造血干细胞标志分子Sca~+-Ⅰ表达。而采用免疫磁珠方法分离Sca~+-Ⅰ细胞，流式检测发现rhIGF-Ⅰ处理后巨核细胞分化标志分子CD41表达水平明显升高，这些结果提示rhIGF-Ⅰ可能具有促进造血干细胞增殖和向巨核细胞分化的作用。 4、CCK-8法检测发现，体外培养的巨核细胞株MO7e给予rhIGF-Ⅰ处理，其吸光度值未出现显著改变，提示rhIGF-Ⅰ可能不会直接促进巨核细胞增殖。 5、流式检测发现，体外分离培养的骨髓单核细胞在受到辐照射前给予rhIGF-Ⅰ处理，细胞凋亡率显著下降；同样，rhIGF-Ⅰ处理也能显著抑制辐照诱导的巨核细胞凋亡，提示rhIGF-Ⅰ对包括巨核细胞在内的骨髓有核细胞具有抗辐射凋亡作用。 6、倒置显微镜观察发现，rhIGF-Ⅰ能够促进晚期成熟巨核细胞形态发生改变，激光共聚焦证实rhIGF-Ⅰ处理可以导致巨核细胞actin和β1-tubulin向细胞一侧聚集，提示rhIGF-Ⅰ可能具有调节巨核细胞内细胞骨架分布与重排、促进血小板前体生成的作用。
[Abstract]:Ionizing radiation damage not only in the explosion of nuclear weapons, nuclear terrorist attacks, caused by natural disaster or man-made factors of nuclear accident, nuclear leakage cases often occur, such as Japan, Hiroshima, Nagasaki and the former Soviet atomic bomb explosion in Chernobyl nuclear power plant accident in the tens of millions of people affected by ionizing radiation damage.
Bone marrow is the radiation sensitive organs, large dose of ionizing radiation can induce the bone marrow hematopoietic dysfunction, including erythroid, myeloid and megakaryocytic hematopoietic cells decreased, and the resulting peripheral red blood cells, white blood cells and platelets were significantly reduced. The human or animal by radiation damage occurs after bleeding, infection and material metabolic disorders and other symptoms, including hematopoietic dysfunction caused by platelet and leukocyte decrease bleeding, infection and the main cause of death. After years of research, people due to bone marrow hematopoietic function obstacle occurrence mechanism has a profound understanding of radiation damage, in addition to ionizing radiation can directly damage the bone marrow nucleated cells, to further reduce the other the factors can also cause hematopoietic cells, such as in the early of radiation injury, burn, Professor Cheng Tianmin I room had observed in rats and dogs The bone marrow megakaryocytes may be neutrophil microbivorous phenomenon, and named it "cell-cell interaction" (megakaryocytophagia). Therefore, the number of megakaryocytes in bone marrow and peripheral blood platelet levels decreased after radiation injury is particularly significant, how to promote bone marrow hematopoietic recovery after radiation injury is especially effective to enhance megakaryocyte and platelet levels, is a key link of radiation injury.
Insulin like growth factor (IGF) by Rinderkencth in 1976 was originally isolated from human serum, IGF has two, respectively, IGF- I and IGF- II of.IGF- gene structure and proinsulin is highly homologous, and has similar insulin functions named.IGF- 1 is a growth regulator the composition consists of a single polypeptide chain of 70 amino acids, the molecular weight of 7.5kD molecule containing 3 disulfide bonds of two.IGF- 1 is an important effector molecule of growth hormone, its formation is mainly affected by the regulation of growth hormone, growth hormone can stimulate the study around the secretion of IGF- 1. Under normal circumstances, the body of the IGF- I mainly by the liver cells, renal cells, spleen cells and other cells to produce autocrine or paracrine, most of IGF- I and insulin-like growth factor binding protein (IGFBP) binding exists in the.IGF- in blood of the Lord Through its receptor R mediated IGF- I play a wide range of biological effects, is an important regulator of cell proliferation. The results indicate that the recombinant human IGF- 1 (rhIGF- 1) not only has the effect of promoting cell proliferation, but also can play the anti apoptotic function of rhIGF-, such as ionizing radiation induced apoptosis of salivary gland cells and intestinal epithelial cells significantly inhibited.
RhIGF- I also play an important role in promoting bone marrow formation reported in the literature, the in vitro rhIGF- I can not only promote hematopoietic CD34~+ umbilical cord blood derived stem cells, and can promote bone marrow erythroid, myeloid, multi generation colony lymphatic system. However, rhIGF- I generated on megakaryocyte it also has regulatory effect, has not been investigated and reported. In addition, whether in vivo rhIGF- I can effectively promote bone marrow hematopoietic function, and has anti radiation induced apoptosis of bone marrow cells, which are also lack of systematic research. On the other hand, in clinic, by, hematopoietic dysfunction caused by chemotherapy and other reasons are more common, how to effectively promote the hematopoietic function of bone marrow recovery has been a hot topic in clinical research. Therefore, in-depth study of rhIGF- after radiation injury of hematopoietic function recovery process Its role, especially its regulation and regulation on megakaryocyte hematopoiesis, not only helps further understand the function of IGF- I, but also provides ideas and basis for the development of new hematopoietic regulatory drugs.
This paper focuses on the research of rhIGF- on radiation injury in mice megakaryocyte hematopoietic function recovery effect and molecular mechanism. BALB/c mice were selected as the research object, first through pathological examination and tissue blood dynamic observation and analysis of peripheral blood cells and bone marrow in irradiated mice given rhIGF- I treated change key nuclear cell counts. To observe the effect of rhIGF- 1 treatment on megakaryocytes and platelet levels in peripheral blood and bone marrow, clear rhIGF- I have to promote radiation injury in mice megakaryocyte hematopoiesis function recovery; bone marrow mononuclear cells and hematopoietic stem cells, the analysis of expression of megakaryocytic differentiation of rhIGF- markers after treatment, observation of rhIGF- I can promote hematopoietic stem cells to differentiate into megakaryocytes; megakaryocytes were further cultured in vitro as the research object, research rhIGF- I The effects of megakaryocyte proliferation and irradiation induced megakaryocyte apoptosis, as well as the distribution and rearrangement of megakaryocyte cytoskeleton actin and 1-tubulin, reveal the role and mechanism of megakaryocyte proliferation in promoting megakaryocyte hematopoietic function recovery.
Conclusion:
1, BALB/c mice were immediately subcutaneously injected with 100 7.5Gy g/kg rhIGF- after being exposed to gamma rays for a whole time. Compared with the irradiated control group, the survival rate of the 40 day survival group increased by nearly 20%, indicating that rhIGF- I had an obvious radiation protection effect.
2, peripheral blood cell counting results showed that received 5.0Gy gamma irradiation in mice, administration of rhIGF- I 10-17 days after treatment, in addition to the number of red blood cells and white blood cells increased significantly, rhIGF- I treated mice peripheral blood platelet levels were significantly higher than those in the control group found that bone marrow irradiation; histopathology rhIGF- I, radiation injury after treatment, the number of mononuclear cells significantly increased includingmegakaryocytes, bone marrow; colony forming experiment showed that rhIGF- of mice were treated with CFU-GM, BFU-E and CFU-GEMM colonies were significantly increased. These results suggest that rhIGF- can promote the synthesis of radiation injury mice including megakaryocyte bone marrow hematopoietic function recovery.
3, I give rhIGF- after irradiation treatment, mouse bone marrow c-kit~+ cells increased significantly, further analysis showed that rhIGF- I can promote hematopoietic stem cell marker expression in Lin~-c-kit~+ cells. Sca~+- I used immunomagnetic separation method of Sca~+- cell, flow cytometry megakaryocyte differentiation is rhIGF- I treated marker CD41 expression the level increased significantly, these results suggest that rhIGF-, I may have a role in promoting the proliferation of hematopoietic stem cells and megakaryocytic differentiation.
4, CCK-8 assay showed that MO7e rhIGF- treated in vitro showed no significant change in absorbance value, suggesting that rhIGF- I might not directly promote the proliferation of megakaryocytes.
5, flow cytometry showed that the cultured bone marrow mononuclear cells treated with rhIGF- irradiation in treatment of shot before in vitro, the apoptosis rate decreased significantly; similarly, megakaryocyte apoptosis of rhIGF- treatment can also significantly inhibit radiation induced, suggesting that rhIGF- of nucleated cells with anti apoptotic effect on radiation includingmegakaryocytes including the bone marrow.
6, found that the inverted microscope was used to observe the rhIGF- I can promote the late mature megakaryocytes morphological changes, confocal laser confirmed rhIGF- I can lead to megakaryocyte actin and beta 1-tubulin gathered to the side of the cell, suggesting that rhIGF- may regulate the cellular skeleton of megakaryocyte in distribution and rearrangement, promote the formation of platelet precursors.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R82

【相似文献】