本文選題：氨磷汀 + 輻射損傷��； 參考：《第二軍醫(yī)大學》2013年碩士論文

【摘要】：目的：研究氨磷汀的抗輻射作用，尤其是對外周血細胞以及骨髓細胞的保護作用，為氨磷汀進一步應用于臨床以及核戰(zhàn)爭抗輻射作用提供依據(jù)。方法：清潔級ICR小鼠，雌雄各半，按體重隨機分為正常組、模型組、氨磷汀低劑量組(120mg/kg)、中劑量組(240mg/kg)、高劑量組(480mg/kg)。5組小鼠連續(xù)給藥3天，第3天在給藥結束15~30分鐘內(nèi)對除正常組以外的其他4組小鼠給予60Coγ射線全身照射，其中30d存活率及存活天數(shù)試驗照射劑量為8Gy，其余試驗照射劑量均為6Gy，然后觀察輻射及氨磷汀對小鼠30d存活率的影響、體重及外周血細胞計數(shù)的影響、氧化應激指標的影響、造血免疫器官的影響。同時取另一批ICR小鼠，雌雄各半，按體重隨機分為正常組、模型組、氨磷汀給藥組(480mg/kg)。3組小鼠連續(xù)給藥3天，第3天在給藥結束15~30min內(nèi)對除正常組以外的其他兩組小鼠給予60Coγ射線全身照射，輻射后6、12、24h取小鼠骨髓細胞進行凋亡測定。結果：(1)30d存活率及存活天數(shù)：低劑量組、中劑量組、高劑量組的平均存活時間與模型組相比均延長，尤其是高劑量組延長小鼠存活時間與模型組比較有統(tǒng)計學意義。(2)體重：輻射后第1天、第4天，模型組、低劑量組、中劑量組、高劑量組體重均下降，且與正常組比較差異有統(tǒng)計學意義；輻射后第7天、第14天，模型組、低劑量組、中劑量組、高劑量組小鼠體重一直呈上升趨勢，僅模型組與正常組小鼠體重的差異有統(tǒng)計學意義；第21天至實驗結束，各組小鼠體重均呈上升趨勢，且差異無統(tǒng)計學意義。(3)血液學指標：輻射后至第7天，模型組、低劑量組、中劑量組、高劑量組小鼠外周血白細胞數(shù)與正常組小鼠相比急劇下降，且差異有統(tǒng)計學意義，而高劑量組外周血白細胞數(shù)比模型組小鼠高，且差異有統(tǒng)計學意義，輻射后第14天，模型組、低劑量組、中劑量組小鼠外周血白細胞數(shù)與正常組小鼠相比仍下降，且差異有統(tǒng)計學意義，而高劑量組小鼠外周血白細胞數(shù)與正常組小鼠的差異已無統(tǒng)計學意義，輻射后第21天開始直至實驗結束，模型組、低劑量組、中劑量組、高劑量組小鼠外周血白細胞數(shù)與正常組小鼠相比差異已無統(tǒng)計學意義；輻射后第1、4、7天，模型組、低劑量組、中劑量組、高劑量組小鼠外周血RBC數(shù)與正常組小鼠相比下降，且差異有統(tǒng)計學意義，輻射后第14天，模型組、低劑量組、中劑量組、高劑量組小鼠外周血紅細胞數(shù)與正常組小鼠相比下降，但只有模型組和低劑量組與正常組的差異有統(tǒng)計學意義，輻射后第21天開始直至實驗結束，模型組、低劑量組、中劑量組、高劑量組小鼠外周血紅細胞數(shù)與正常組小鼠相比差異已無統(tǒng)計學意義；輻射后第1、4、7天，模型組、低劑量組、中劑量組、高劑量組小鼠外周血淋巴細胞數(shù)與正常組小鼠相比下降，且差異有統(tǒng)計學意義，而高劑量組外周血淋巴細胞數(shù)比模型組小鼠高，且差異有統(tǒng)計學意義，輻射后第14天，模型組、低劑量組、中劑量組小鼠外周血淋巴細胞數(shù)與正常組小鼠相比仍下降，且差異有統(tǒng)計學意義，輻射后第28天，所有小鼠外周血淋巴細胞數(shù)與正常組小鼠相比差異已無統(tǒng)計學意義；輻射后第14天，模型組、低劑量組小鼠外周血小板數(shù)與正常組小鼠相比急劇下降，且差異有統(tǒng)計學意義，而中劑量組和高劑量組小鼠外周血小板數(shù)與正常組小鼠的差異無統(tǒng)計學意義。(4)氧化應激指標：輻射后第3天，模型組、低劑量組、中劑量組、高劑量組小鼠的SOD活力均比正常組小鼠的SOD活力降低，且差異有統(tǒng)計學意義；而中劑量組和高劑量組小鼠的SOD活力比模型組小鼠的SOD活力升高，且差異有統(tǒng)計學意義；輻射后第7天，模型組、低劑量組小鼠的MDA含量均比正常組小鼠的MDA含量升高，且差異有統(tǒng)計學意義，而中劑量組和高劑量組小鼠的MDA含量比模型組小鼠的MDA含量降低，差異有統(tǒng)計學意義，而與正常組小鼠的MDA含量相比無差異。(5) TI與SI：輻射后第3天，模型組、低劑量組、中劑量組、高劑量組小鼠的TI和SI與正常組小鼠相比均下降，且差異有統(tǒng)計學意義，而低劑量組、中劑量組、高劑量組小鼠的TI和SI與模型組相比均升高，且差異有統(tǒng)計學意義。(6)胸腺、脾臟與骨髓組織病理學檢查：模型組和給藥組胸腺皮質(zhì)淋巴細胞均減少，胸腺萎縮，，與正常組差異顯著。但各個劑量組以及模型組之間未見明顯差異；組織病理學鏡下觀察各組小鼠脾臟組織可見：模型組與正常組對比，淋巴細胞減少，脾臟造血細胞基本缺如，紅細胞數(shù)目上升。給藥組與模型組對比，淋巴細胞數(shù)目增多，造血細胞數(shù)量相對升高，脾小體數(shù)目和體積均增多、增大，且有著量-效依賴關系；組織病理學鏡下觀察各組小鼠骨髓組織可見：模型組骨髓內(nèi)造血細胞減少，成熟紅細胞大量填充，造血功能低下。給藥組與模型組對比，骨髓腔內(nèi)造血細胞較多，造血功能較強，且有著量-效依賴關系。(7)細胞凋亡：小鼠受輻射后24h內(nèi)細胞凋亡發(fā)生率逐漸升高，模型組小鼠各時間點的骨髓細胞凋亡率與正常組相比均明顯升高，且差異有統(tǒng)計學意義；而氨磷汀可明顯抑制輻射造成的小鼠骨髓細胞凋亡率的升高，且與模型組相比，差異有統(tǒng)計學意義。結論：氨磷汀可提高受輻射小鼠的存活率，延長存活時間；促進受輻射小鼠外周血白細胞、紅細胞、淋巴細胞和血小板的恢復；提高TI和SI；抑制脾臟和骨髓組織的損傷；增強受輻射小鼠機體的SOD活力，降低MDA水平；明顯抑制輻射造成的小鼠骨髓細胞凋亡率的升高，因此氨磷汀對輻射后小鼠在血液、抗氧化、免疫和造血四個方面，均顯示出一定的保護和恢復作用。 目的：利用HL-60人原髓細胞白血病細胞系，K562人慢性髓原白血病細胞系以及免疫磁珠分選(Magnetic activated cell sorting, MACS)技術獲得的骨髓造血干/祖細胞進一步探討輻射造成骨髓損傷的原因與機制，輻射誘導細胞凋亡是否存在特有的分子機制以及氨磷汀保護受輻射骨髓細胞的機制。方法：HL-60細胞、K562細胞給予不同劑量的氨磷汀后15~30min內(nèi)進行60Coγ射線照射，照射劑量為6Gy，24h后對HL-60細胞、K562細胞進行凋亡測定；30只小鼠，雌雄各半，按體重隨機分為正常組、模型組、氨磷汀給藥組(480mg/kg)，3組小鼠連續(xù)給藥3天，第3天在給藥結束15~30分鐘內(nèi)對除正常組以外的其他兩組小鼠給予60Coγ射線全身照射，輻射后24h取骨髓細胞進行CD117+細胞計數(shù)，并利用MACS法分選骨髓造血干/祖細胞，對其進行凋亡測定，并用Western Blot方法對骨髓造血干/祖細胞中的p53、p21、mdm2及c-myc基因所表達的蛋白進行定量。結果：(1)輻射后24h，HL-60細胞隨著氨磷汀給藥量的增加，其凋亡率較正常組也逐漸增加。(2)輻射后24h，K562細胞隨著氨磷汀給藥量的增加，其凋亡率較正常組也逐漸增加。(3)小鼠輻射后24h后，對骨髓細胞進行CD117抗體標記，得到骨髓中CD117+細胞的百分比，模型組小鼠骨髓細胞中CD117+細胞的百分比與正常組相比明顯下降，且差異有統(tǒng)計學意義，而氨磷汀給藥小鼠骨髓細胞中CD117+細胞的百分比與模型組相比有所上升，且差異有統(tǒng)計學意義。(4)小鼠受輻射后24h，模型組小鼠的骨髓造血干/祖細胞凋亡率與正常組相比均明顯升高，且差異有統(tǒng)計學意義；而氨磷汀給藥組可抑制輻射造成的小鼠骨髓細胞凋亡率的升高，且與模型組相比，差異有統(tǒng)計學意義。(5)利用Western Blot方法對基因p53、p21、mdm2及c-myc所表達的蛋白進行定量發(fā)現(xiàn)氨磷汀能下調(diào)造血干/祖細胞中的p21及c-myc表達，上調(diào)mdm2的表達。結論：(1)氨磷汀能夠影響正常骨髓細胞的p53相關的基因，如p21、mdm2及c-myc等，使這些基因相互調(diào)節(jié)，從而起到抑制細胞凋亡，保護骨髓細胞的作用。(2)氨磷汀對凋亡具有雙重作用，而這種作用可能與細胞的種類有關。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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