氨磷汀的抗辐射损伤作用及其机制研究

发布时间：2018-05-26 14:05

本文选题：氨磷汀 + 辐射损伤　；参考：《第二军医大学》2013年硕士论文

【摘要】：目的：研究氨磷汀的抗辐射作用，尤其是对外周血细胞以及骨髓细胞的保护作用，为氨磷汀进一步应用于临床以及核战争抗辐射作用提供依据。方法：清洁级ICR小鼠，雌雄各半，按体重随机分为正常组、模型组、氨磷汀低剂量组(120mg/kg)、中剂量组(240mg/kg)、高剂量组(480mg/kg)。5组小鼠连续给药3天，第3天在给药结束15~30分钟内对除正常组以外的其他4组小鼠给予60Coγ射线全身照射，其中30d存活率及存活天数试验照射剂量为8Gy，其余试验照射剂量均为6Gy，然后观察辐射及氨磷汀对小鼠30d存活率的影响、体重及外周血细胞计数的影响、氧化应激指标的影响、造血免疫器官的影响。同时取另一批ICR小鼠，雌雄各半，按体重随机分为正常组、模型组、氨磷汀给药组(480mg/kg)。3组小鼠连续给药3天，第3天在给药结束15~30min内对除正常组以外的其他两组小鼠给予60Coγ射线全身照射，辐射后6、12、24h取小鼠骨髓细胞进行凋亡测定。结果：(1)30d存活率及存活天数：低剂量组、中剂量组、高剂量组的平均存活时间与模型组相比均延长，尤其是高剂量组延长小鼠存活时间与模型组比较有统计学意义。(2)体重：辐射后第1天、第4天，模型组、低剂量组、中剂量组、高剂量组体重均下降，且与正常组比较差异有统计学意义；辐射后第7天、第14天，模型组、低剂量组、中剂量组、高剂量组小鼠体重一直呈上升趋势，仅模型组与正常组小鼠体重的差异有统计学意义；第21天至实验结束，各组小鼠体重均呈上升趋势，且差异无统计学意义。(3)血液学指标：辐射后至第7天，模型组、低剂量组、中剂量组、高剂量组小鼠外周血白细胞数与正常组小鼠相比急剧下降，且差异有统计学意义，而高剂量组外周血白细胞数比模型组小鼠高，且差异有统计学意义，辐射后第14天，模型组、低剂量组、中剂量组小鼠外周血白细胞数与正常组小鼠相比仍下降，且差异有统计学意义，而高剂量组小鼠外周血白细胞数与正常组小鼠的差异已无统计学意义，辐射后第21天开始直至实验结束，模型组、低剂量组、中剂量组、高剂量组小鼠外周血白细胞数与正常组小鼠相比差异已无统计学意义；辐射后第1、4、7天，模型组、低剂量组、中剂量组、高剂量组小鼠外周血RBC数与正常组小鼠相比下降，且差异有统计学意义，辐射后第14天，模型组、低剂量组、中剂量组、高剂量组小鼠外周血红细胞数与正常组小鼠相比下降，但只有模型组和低剂量组与正常组的差异有统计学意义，辐射后第21天开始直至实验结束，模型组、低剂量组、中剂量组、高剂量组小鼠外周血红细胞数与正常组小鼠相比差异已无统计学意义；辐射后第1、4、7天，模型组、低剂量组、中剂量组、高剂量组小鼠外周血淋巴细胞数与正常组小鼠相比下降，且差异有统计学意义，而高剂量组外周血淋巴细胞数比模型组小鼠高，且差异有统计学意义，辐射后第14天，模型组、低剂量组、中剂量组小鼠外周血淋巴细胞数与正常组小鼠相比仍下降，且差异有统计学意义，辐射后第28天，所有小鼠外周血淋巴细胞数与正常组小鼠相比差异已无统计学意义；辐射后第14天，模型组、低剂量组小鼠外周血小板数与正常组小鼠相比急剧下降，且差异有统计学意义，而中剂量组和高剂量组小鼠外周血小板数与正常组小鼠的差异无统计学意义。(4)氧化应激指标：辐射后第3天，模型组、低剂量组、中剂量组、高剂量组小鼠的SOD活力均比正常组小鼠的SOD活力降低，且差异有统计学意义；而中剂量组和高剂量组小鼠的SOD活力比模型组小鼠的SOD活力升高，且差异有统计学意义；辐射后第7天，模型组、低剂量组小鼠的MDA含量均比正常组小鼠的MDA含量升高，且差异有统计学意义，而中剂量组和高剂量组小鼠的MDA含量比模型组小鼠的MDA含量降低，差异有统计学意义，而与正常组小鼠的MDA含量相比无差异。(5) TI与SI：辐射后第3天，模型组、低剂量组、中剂量组、高剂量组小鼠的TI和SI与正常组小鼠相比均下降，且差异有统计学意义，而低剂量组、中剂量组、高剂量组小鼠的TI和SI与模型组相比均升高，且差异有统计学意义。(6)胸腺、脾脏与骨髓组织病理学检查：模型组和给药组胸腺皮质淋巴细胞均减少，胸腺萎缩，，与正常组差异显著。但各个剂量组以及模型组之间未见明显差异；组织病理学镜下观察各组小鼠脾脏组织可见：模型组与正常组对比，淋巴细胞减少，脾脏造血细胞基本缺如，红细胞数目上升。给药组与模型组对比，淋巴细胞数目增多，造血细胞数量相对升高，脾小体数目和体积均增多、增大，且有着量-效依赖关系；组织病理学镜下观察各组小鼠骨髓组织可见：模型组骨髓内造血细胞减少，成熟红细胞大量填充，造血功能低下。给药组与模型组对比，骨髓腔内造血细胞较多，造血功能较强，且有着量-效依赖关系。(7)细胞凋亡：小鼠受辐射后24h内细胞凋亡发生率逐渐升高，模型组小鼠各时间点的骨髓细胞凋亡率与正常组相比均明显升高，且差异有统计学意义；而氨磷汀可明显抑制辐射造成的小鼠骨髓细胞凋亡率的升高，且与模型组相比，差异有统计学意义。结论：氨磷汀可提高受辐射小鼠的存活率，延长存活时间；促进受辐射小鼠外周血白细胞、红细胞、淋巴细胞和血小板的恢复；提高TI和SI；抑制脾脏和骨髓组织的损伤；增强受辐射小鼠机体的SOD活力，降低MDA水平；明显抑制辐射造成的小鼠骨髓细胞凋亡率的升高，因此氨磷汀对辐射后小鼠在血液、抗氧化、免疫和造血四个方面，均显示出一定的保护和恢复作用。目的：利用HL-60人原髓细胞白血病细胞系，K562人慢性髓原白血病细胞系以及免疫磁珠分选(Magnetic activated cell sorting, MACS)技术获得的骨髓造血干/祖细胞进一步探讨辐射造成骨髓损伤的原因与机制，辐射诱导细胞凋亡是否存在特有的分子机制以及氨磷汀保护受辐射骨髓细胞的机制。方法：HL-60细胞、K562细胞给予不同剂量的氨磷汀后15~30min内进行60Coγ射线照射，照射剂量为6Gy，24h后对HL-60细胞、K562细胞进行凋亡测定；30只小鼠，雌雄各半，按体重随机分为正常组、模型组、氨磷汀给药组(480mg/kg)，3组小鼠连续给药3天，第3天在给药结束15~30分钟内对除正常组以外的其他两组小鼠给予60Coγ射线全身照射，辐射后24h取骨髓细胞进行CD117+细胞计数，并利用MACS法分选骨髓造血干/祖细胞，对其进行凋亡测定，并用Western Blot方法对骨髓造血干/祖细胞中的p53、p21、mdm2及c-myc基因所表达的蛋白进行定量。结果：(1)辐射后24h，HL-60细胞随着氨磷汀给药量的增加，其凋亡率较正常组也逐渐增加。(2)辐射后24h，K562细胞随着氨磷汀给药量的增加，其凋亡率较正常组也逐渐增加。(3)小鼠辐射后24h后，对骨髓细胞进行CD117抗体标记，得到骨髓中CD117+细胞的百分比，模型组小鼠骨髓细胞中CD117+细胞的百分比与正常组相比明显下降，且差异有统计学意义，而氨磷汀给药小鼠骨髓细胞中CD117+细胞的百分比与模型组相比有所上升，且差异有统计学意义。(4)小鼠受辐射后24h，模型组小鼠的骨髓造血干/祖细胞凋亡率与正常组相比均明显升高，且差异有统计学意义；而氨磷汀给药组可抑制辐射造成的小鼠骨髓细胞凋亡率的升高，且与模型组相比，差异有统计学意义。(5)利用Western Blot方法对基因p53、p21、mdm2及c-myc所表达的蛋白进行定量发现氨磷汀能下调造血干/祖细胞中的p21及c-myc表达，上调mdm2的表达。结论：(1)氨磷汀能够影响正常骨髓细胞的p53相关的基因，如p21、mdm2及c-myc等，使这些基因相互调节，从而起到抑制细胞凋亡，保护骨髓细胞的作用。(2)氨磷汀对凋亡具有双重作用，而这种作用可能与细胞的种类有关。
[Abstract]:Objective: To study the anti radiation effect of ammoniacine, especially the protective effect of peripheral blood cells and bone marrow cells, and to provide the basis for the further application of ammoniacine in clinical and nuclear war radiation resistance. Methods: clean grade ICR mice were divided into normal group, model group and low dose group of ammoniphosphine (120mg/kg). In the medium dose group (240mg/kg), the mice in the high dose group (480mg/kg).5 group were given the drug for 3 days, and the third day was given the whole body irradiation to the other 4 groups of mice except the normal group at the end of the third day. The 30d survival rate and the survival days were 8Gy, the rest of the experimental irradiation dose were 6Gy, and then the radiation and ammonia and phosphorus were observed. The effect of tine on the survival rate of 30d in mice, the influence of weight and peripheral blood cell count, the influence of oxidative stress index and the influence of hematopoietic immune organs. At the same time, another group of ICR mice were divided into normal group, the model group, the amamatostine administration group (480mg/kg) group.3 mice were given 3 days, and the third days were given 15~30min. Two groups of mice other than the normal group were irradiated with 60Co gamma ray. The apoptosis of mouse bone marrow cells was measured by 6,12,24h after radiation. Results: (1) the survival rate of 30d and the number of survival days: the average survival time of the low dose group, middle dose group and high dose group prolonged, especially in the high dose group. The living time was statistically significant compared with the model group. (2) weight: first days after radiation, fourth days after radiation, the model group, low dose group, middle dose group, high dose group decreased, and the difference was statistically significant compared with the normal group; seventh days, fourteenth days after radiation, the model group, low dose group, middle dose group, and high dose group had been on the rise in body weight. The weight difference between the model group and the normal group was statistically significant. Twenty-first days to the end of the experiment, the weight of the mice in each group showed an upward trend, and the difference was not statistically significant. (3) the Hematology Index: after radiation to seventh days, the model group, low dose group, middle dose group, and high dose group of mice were compared with normal mice. The number of white blood cells in the peripheral blood of the high dose group was higher than that of the model group, and the difference was statistically significant. In the fourteenth day after radiation, the number of white blood cells in the peripheral blood of the model group, the low dose group and the middle dose group was still lower than that of the normal group, and the difference was statistically significant, but the high dose group of the peripheral blood of the mice. There was no significant difference between the number of white blood cells and the normal group. The difference between the model group, the low dose group, the middle dose group, the high dose group and the normal group was no significant difference between the model group, the low dose group, the middle dose group, the high dose group and the normal group, and the model group, the low dose group, the middle dose group, the high dose after radiation were 1,4,7 days after radiation. The number of peripheral blood RBC in the group of mice was lower than that in the normal group, and the difference was statistically significant. The number of peripheral blood erythrocytes in the model group, the low dose group, the middle dose group, the high dose group and the normal group decreased at fourteenth days after radiation, but the difference between the model group and the low dose group was statistically significant, twenty-first days after radiation. The number of peripheral blood erythrocytes in the model group, the low dose group, the middle dose group and the high dose group had no significant difference compared with the normal group. The number of peripheral blood lymphocytes in the model group, the low dose group, the low dose group, the middle dose group and the high dose group decreased compared with the normal group after 1,4,7 day after radiation. The number of peripheral blood lymphocytes in the high dose group was higher than that of the model group, and the difference was statistically significant. The number of lymphocytes in the peripheral blood of the mice in the model group, the low dose group, the middle dose group and the normal group decreased at fourteenth days after radiation, and the difference was statistically significant, and the peripheral blood lymphocytes of all mice were twenty-eighth days after radiation. There was no significant difference in the number compared with the normal group. After fourteenth days of radiation, the number of peripheral platelets in the model group and the low dose group decreased sharply compared with the normal mice, and the difference was statistically significant. There was no significant difference in the number of peripheral platelets from the normal group in the middle dose group and the high dose group. (4) the oxidation should be of no significant difference. Third days after radiation, the SOD activity of mice in the model group, the low dose group, the middle dose group and the high dose group was lower than that of the normal group, and the difference was statistically significant, while the SOD activity of the middle dose group and the high dose group was higher than that of the model group, and the difference was statistically significant, and the difference was statistically significant. Seventh days after the radiation, the mice of the high dose group and the high dose group had a significant difference. In the model group, the content of MDA in the low dose group was higher than that of the normal group, and the difference was statistically significant, but the content of MDA in the middle dose group and the high dose group was lower than that of the model mice. The difference was statistically significant, but there was no difference compared with the MDA content in the normal mice. (5) TI and SI: third days after radiation, Model group, low dose group, medium dose group and high dose group, TI and SI were decreased compared with normal group, and the difference was statistically significant, while low dose group, middle dose group, high dose group, TI and SI in high dose group were higher than model group, and the difference was statistically significant. (6) pathological examination of thymus, spleen and bone marrow tissue: model group The thymus cortical lymphocytes decreased and the thymus atrophy was significantly different from the normal group, but there was no significant difference between the various dose groups and the model group. The spleen tissues of each group were observed under the histopathological microscope: the model group was compared with the normal group, the decrease of lymphocyte, the basic absence of the spleen hematopoietic cells, the number of red blood cells. Compared with the model group, the number of lymphocytes increased, the number of hematopoietic cells increased relatively, the number and volume of spleen corpuscles increased and increased, and there was a dose effect dependence. The bone marrow tissue of mice in each group was observed under the histopathology microscope: the bone marrow cells in the model group were reduced, the mature red blood cells were filled and the hematopoiesis worked. Compared with the model group, there were more hematopoietic cells and more hematopoietic function in the bone marrow cavity, and there was a dose effect dependence. (7) apoptosis: the apoptosis rate of cells in 24h increased gradually in mice after radiation, and the apoptosis rate of bone marrow cells in each time point of the model group was significantly higher than that in the normal group, and the difference was statistically significant. It could significantly inhibit the increase of apoptosis rate of bone marrow cells in mice induced by radiation, and compared with the model group, the difference was statistically significant. Conclusion: it can improve the survival rate of irradiated mice, prolong the survival time, and promote the recovery of white cells, red blood cells, lymphocytes and platelets in the peripheral blood of irradiated mice. Increase TI and SI, inhibit the injury of spleen and bone marrow tissue, enhance the SOD activity of the irradiated mice, reduce the level of MDA, and obviously inhibit the increase of apoptosis rate of bone marrow cells in mice caused by radiation, so amifostine showed certain protection and recovery in four aspects of blood, antioxidant, immunity and hematopoiesis in irradiated mice. Use.
Objective: to further explore the causes and mechanisms of bone marrow damage caused by radiation from HL-60 human myelocytic leukemia cell lines, K562 human chronic myelogenous leukemia cell lines and Magnetic activated cell sorting (MACS) technology, and whether radiation induced apoptosis is unique. The mechanism of molecular mechanism and the protective effect of ammoniacine on the irradiated bone marrow cells. Methods: HL-60 cells, K562 cells were irradiated with 60Co gamma ray in 15~30min after different doses of ammoniacine. The dose of irradiation was 6Gy, 24h and HL-60 cells and K562 cells were measured after 24h; 30 mice were divided into normal groups according to weight and the model was divided into normal group, and the model was divided into normal group and model. Group (480mg/kg), 3 groups of mice were administered for 3 days. On the third day, the whole body was irradiated with 60Co gamma ray in the other two groups of mice except the normal group in 15~30 minutes. The CD117+ cells were counted by 24h after radiation, and the bone marrow hematopoietic stem / progenitor cells were selected by MACS method. The Western Blot method was used to quantify the protein expressed by p53, p21, MDM2 and c-myc genes in bone marrow hematopoietic stem / progenitor cells. Results: (1) after radiation, the apoptosis rate of 24h and HL-60 cells increased gradually with the increase of the dosage of ammoniacine. (2) 24h after radiation, the apoptosis rate of K562 cells was compared with the increase of the dosage of ammoniacine. The normal group also increased gradually. (3) after the mice were irradiated after 24h, the bone marrow cells were labeled with CD117 antibody, and the percentage of CD117+ cells in the bone marrow was obtained. The percentage of CD117+ cells in the bone marrow cells in the model group was significantly lower than that in the normal group, and the difference was statistically significant, and the CD117+ cells in the bone marrow cells of the mice were given 100% of the CD117+ cells. Compared with the model group, the difference was statistically significant. (4) the apoptosis rate of bone marrow hematopoietic stem / progenitor cells in the model mice was significantly higher than that in the normal group after radiation 24h, and the difference was statistically significant. The difference was statistically significant. (5) the Western Blot method was used to quantify the expression of p21 and c-myc in hematopoietic stem / progenitor cells and up regulation of the expression of p21 and c-myc in gene p53, p21, MDM2 and c-myc. Conclusion: (1) p53 related genes of normal bone marrow cells can be affected by ammoniacine, such as p21, MDM2, and vegetables. -myc and other genes regulate each other to inhibit apoptosis and protect the role of bone marrow cells. (2) amiostine has a double effect on apoptosis, and this effect may be related to the type of cell.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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