电离辐射引起骨髓微环境中趋化因子CCL3及CXCL14浓度改变机制

发布时间：2018-05-26 15:38

本文选题：电离辐射 + 趋化因子　；参考：《上海交通大学》2013年硕士论文

【摘要】：电离辐射会引起机体基因的改变从而引发一系列病理生理变化，因此从这一变化的机制角度加以分析可以为辐射防护研究以及辐射相关的药物开发提供参考。在对前期基因芯片结果进行进一步分析后，我们得到了在辐照导致的骨髓损伤的修复阶段，免疫反应（主要是造血作用）发挥了关键性功能；主要参与造血干细胞的自我更新、增殖及分化，加速机体恢复。因此，我们选择参与免疫反应（包括造血作用）的分泌性趋化因子CCL3和CXCL14进行蛋白水平的初步分析。并且构建假说：在辐射损伤修复阶段，机体通过自发下调CCL3表达以及增加CCL3的水解作用促进干细胞的增殖，同时上调CXCL14的表达促进机体修复。首先，构建半致死剂量辐照损伤动物模型，通过外周血细胞及骨髓有核细胞计数以及骨髓细胞涂片观察在损伤修复阶段血细胞的改变及恢复情况。其次，通过ELISA试剂盒对辐照损伤动物模型骨髓微环境中CCL3和CXCL14蛋白表达水平进行检测，并对骨髓细胞分泌的能够水解CCL3的蛋白水解酶CtsG蛋白含量通过Western Blot方法进行检测，分析目标蛋白在辐照损伤修复阶段的浓度的变化趋势以及对于引起这一浓度改变的机制进行探讨。结果显示骨髓损伤修复阶段骨髓有核细胞的恢复先于外周血白细胞及血小板的恢复，红细胞受辐照刺激影响较小；骨髓细胞涂片结果进一步证实了辐照引起了骨髓细胞的严重损伤，其中电离辐射对成熟红细胞的损伤作用最小；蛋白检测结果中骨髓损伤修复过程中微环境中趋化因子CCL3显著降低，CXCL14保持不变；水解CCL3的蛋白水解酶CtsG在细胞中浓度升高。该结果进一步证明CCL3在辐照损伤修复阶段发挥主要作用，机体通过自发下调CCL3及加速其蛋白水解两方面减少CCL3对造血干细胞的增殖抑制。而CXCL14蛋白变化趋势与mRNA不符可能与辐射急性期泛素化水平升高有关，，结果说明CXCL14与辐照损伤后机体恢复无关，然而却可能与电离辐射初期泛素化水平显著升高及电离辐射引起的致癌性有关。综上，我们对前期基因芯片结果进行进一步研究，证明CCL3是机体损伤修复阶段的重要趋化因子蛋白，并得到CXCL14与修复阶段造血作用无关的结论，为之后辐射防护及药物开发提供一定的理论基础。
[Abstract]:Ionizing radiation can cause a series of pathophysiological changes, so the analysis from the perspective of the mechanism can provide a reference for the research of radiation protection and the development of radiation-related drugs. After further analysis of the gene chip results, we found that immune response (mainly hematopoiesis) plays a key role in the repair of bone marrow injury induced by irradiation, mainly involved in the self-renewal of hematopoietic stem cells. Proliferation and differentiation to accelerate the recovery of the body. Therefore, we selected secretory chemokines (CCL3 and CXCL14) involved in immune response (including hematopoiesis) for preliminary analysis of protein levels. The hypothesis was constructed that during the repair stage of radiation injury, the body promoted the proliferation of stem cells through spontaneous down-regulation of CCL3 expression and increased hydrolysis of CCL3, and up-regulated the expression of CXCL14 to promote the repair of organism. Firstly, the animal model of half-lethal dose irradiation injury was established. The changes and recovery of blood cells in the stage of injury and repair were observed by counting peripheral blood cells and bone marrow nucleated cells as well as bone marrow smear. Secondly, the expression levels of CCL3 and CXCL14 protein in bone marrow microenvironment of irradiated animal model were detected by ELISA kit, and the protein content of proteolytic enzyme CtsG which could hydrolyze CCL3 was detected by Western Blot method. The change trend of target protein concentration in the repair phase of irradiation damage and the mechanism of the concentration change were analyzed. The results showed that the recovery of bone marrow nucleated cells was prior to the recovery of peripheral white blood cells and platelets in the stage of bone marrow injury and repair, and the red blood cells were less affected by irradiation stimulation. The results of bone marrow cell smear further confirmed that irradiation caused serious damage to bone marrow cells, in which ionizing radiation had the least effect on mature erythrocytes. In the process of bone marrow injury and repair, the chemokine CCL3 significantly decreased the CXCL14 remained unchanged, while the concentration of proteolytic enzyme CtsG of CCL3 increased in the cells. The results further demonstrated that CCL3 plays a major role in the repair of irradiation injury, and the inhibition of CCL3 on the proliferation of hematopoietic stem cells was reduced by spontaneous down-regulation of CCL3 and accelerated protein hydrolysis of CCL3. However, the change trend of CXCL14 protein and mRNA may be related to the increase of Ubiquitin level in acute radiation stage. The results indicate that CXCL14 is not related to the recovery of body after irradiation. However, it may be associated with a significant increase in the level of ubiquialization in the initial stage of ionizing radiation and carcinogenicity caused by ionizing radiation. In conclusion, we further studied the results of gene microarray in the early stage, which proved that CCL3 is an important chemokine protein in the repair stage of body injury, and it is concluded that CXCL14 has no relationship with hematopoiesis in the repair stage. It provides a theoretical basis for radiation protection and drug development.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R14

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