氡暴露对大鼠免疫系统的毒作用及其机制探讨

发布时间：2018-07-28 12:44

【摘要】：目的：目前国内外对氡及其子体的健康影响主要集中在其致癌作用,尤其是致肺癌作用的研究,而关于氡暴露对免疫系统毒性影响的研究较少。本课题通过观察氡暴露对Wistar大鼠胸腺、脾脏组织及外周血、肺泡灌洗液以及骨髓细胞的损伤作用,探讨氡对大鼠免疫功能的毒作用,为探索氡致机体免疫损伤的可能机制提供实验资料。方法：选用健康雄性Wistar大鼠15只,体重100±10g,随机分成三组,包括对照组、200工作水平月(WLM)组和400工作水平月组,每组5只。除对照组外,其余2组整体暴露于HD-3型多功能生态氡室,累积受照剂量分别达200和400WLM,剂量率保持恒定。大鼠麻醉后,腹主动脉采血,收集支气管肺泡灌洗液(BALF),胸腺、脾脏称重后制成单细胞悬液,分离股骨取骨髓。计算胸腺、脾脏脏器指数,CELL-DYN3700全自动五分类血液分析仪检测BALF、骨髓、外周血细胞计数及分类,应用流式细胞仪技术(FCM)和转荧光质粒细菌检测肺泡巨噬细胞吞噬功能。取制备好的BALF、骨髓、外周血、胸腺和脾脏的单细胞悬液,通过相应的荧光探针检测不同剂量氡暴露组淋巴细胞及粒细胞内活性氧(ROS)、游离钙离子([Ca2+]i)浓度、线粒体膜电位(MMP)水平的变化；通过碘化丙啶(PI)染色观察不同氡暴露组间脾脏、胸腺淋巴细胞凋亡率。结果：(1)与对照组比较,200WLM氡暴露组胸腺指数明显降低,而脾指数无明显改变。大鼠外周血白细胞、中性粒细胞、淋巴细胞、嗜酸性粒细胞、嗜碱性粒细胞、红细胞计数、血红蛋白量、红细胞压积、平均血红蛋白浓度及红细胞分布宽度均明显增加,而红细胞平均体积明显下降。400WLM氡暴露组红细胞计数、血红蛋白含量、红细胞分布宽度明显增加,而红细胞平均体积、平均血红蛋白量显著降低。200WLM和400WLM氡暴露组单核细胞计数、血小板计数与对照组比较,均无显著差异。氡暴露大鼠肺泡灌洗液淋巴细胞、中性粒细胞数量显著增加,骨髓细胞总数、淋巴细胞及中性粒细胞计数均显著增加。(2)200WLM氡暴露组巨噬细胞的吞噬率和吞噬指数显著高于对照组,而400WLM氡暴露组肺泡巨噬细胞吞噬率无明显改变,但吞噬指数显著降低。(3)200WLM氡暴露组胸腺淋巴细胞ROS明显增多,400WLM氡暴露组大鼠肺泡灌洗液粒细胞中的ROS较对照组显著增加。(4)200WLM氡暴露组骨髓、外周血、肺泡灌洗液中淋巴细胞的线粒体膜电位(MMP)较对照组明显降低,而400WLM组仅肺泡灌洗液中的淋巴细胞MMP显著降低。(5)与对照组比较,200WLM组脾、胸腺淋巴细胞和肺泡灌洗液粒细胞钙离子浓度明显升高,而400WLM组肺泡灌洗液淋巴细胞的钙离子浓度明显升高,粒细胞钙离子浓度则明显下降。(6)细胞周期分析发现,200WLM氡暴露组胸腺细胞G0/G1期细胞比例明显降低,而S期细胞比例显著升高,脾细胞则相反。(7)细胞凋亡检测结果显示,200WLM氡暴露组胸腺细胞及400WLM组骨髓细胞凋亡率明显高于对照组,脾和肺泡灌洗细胞的凋亡率未见明显改变。结论：大鼠氡暴露后胸腺指数的减小,淋巴细胞和中性粒细胞数量的改变以及巨噬细胞吞噬功能的降低,表明氡对大鼠免疫细胞和免疫功能产生了毒性作用,而氡染毒后细胞内活性氧、游离钙离子浓度、线粒体膜电位和细胞凋亡率的改变,提示氡及其子体可能是通过氧化损伤和细胞周期障碍等途径影响机体的免疫功能。
[Abstract]:Objective: the health effects of radon and its daughters at home and abroad are mainly concentrated on the carcinogenic effect, especially the effect of lung cancer, and there are few studies on the effects of radon exposure on the immune system toxicity. This topic has been observed by observing radon exposure on the thymus, spleen and peripheral blood, alveolar lavage fluid and bone marrow cells in Wistar rats. To investigate the toxic effects of radon on immune function in rats, and to provide experimental data for exploring the possible mechanism of radon induced immune injury.
Methods: 15 healthy male Wistar rats, weighing 100 + 10g, were randomly divided into three groups, including the control group, the 200 working level month (WLM) group and the 400 working level month group, 5 in each group. Except the control group, the other 2 groups were exposed to the HD-3 multi-functional ecological radon room with the cumulative exposure dose of 200 and 400WLM, and the dose rate kept constant. Rat anesthesia was kept constant. After drunken, the abdominal aorta took blood, collected the bronchoalveolar lavage fluid (BALF), the thymus and the spleen to make a single cell suspension, separated the bone marrow from the femur, calculated the thymus, the spleen visceral index, and measured the BALF, the marrow, the peripheral blood cell count and the classification by the CELL-DYN3700 automatic five classification hematology analyzer. The flow cytometry (FCM) and the transfer of fluorescence were used. The phagocytosis of alveolar macrophages was detected by plasmid bacteria. The single cell suspensions of BALF, bone marrow, peripheral blood, thymus and spleen were prepared. The changes of ROS, [Ca2 +]i concentration, and mitochondrial membrane potential (MMP) level of the lymphocytes and granulocytes in different doses of radon exposure group were detected by the corresponding fluorescence probe. The apoptotic rate of spleen and thymus lymphocytes was observed by propidium iodide (PI) staining.
Results: (1) compared with the control group, the thymus index of 200WLM radon exposure group was significantly lower, but the spleen index was not significantly changed. The peripheral blood leukocyte, neutrophils, lymphocyte, eosinophil, basophil, red cell count, hemoglobin, red cell pressure product, mean hemoglobin concentration and red cell distribution width were all obvious in the rats. The average volume of red blood cells decreased significantly in.400WLM radon exposure group, hemoglobin content, red blood cell distribution width increased significantly, while the average volume of red blood cells, the average hemoglobin volume decreased significantly in.200WLM and 400WLM radon exposure group, and there was no significant difference between the blood plate count and the control group. The number of neutrophils increased significantly, the number of bone marrow cells, lymphocyte and neutrophils increased significantly. (2) the phagocytic rate and phagocytic index of macrophages in 200WLM radon exposure group were significantly higher than those in the control group, but the phagocytosis rate of alveolar macrophages in the 400WLM radon exposed group was not significantly changed, but the phagocytic index of the phagocytosis was not significantly changed. (3) the thymus lymphocyte ROS in 200WLM radon exposure group increased significantly, and the ROS in the granulocyte of alveolar lavage fluid in 400WLM radon exposure group was significantly higher than that of the control group. (4) the mitochondrial membrane potential (MMP) of the lymphocytes in the 200WLM radon exposure group was significantly lower than that in the control group, while the 400WLM group only alveolar lavage. The lymphocyte MMP in the liquid decreased significantly. (5) compared with the control group, the concentration of calcium ions in the spleen, thymus lymphocyte and alveolar lavage fluid in the 200WLM group was significantly increased, while the concentration of calcium ion in the lymphocyte of the alveolar lavage fluid in the 400WLM group was significantly increased and the concentration of granulocyte calcium ion decreased significantly. (6) the cell cycle analysis found that the 200WLM radon exposure was found. The proportion of G0/G1 phase cells in the thymus cells decreased significantly, while the proportion of S cells increased significantly and the spleen cells were the opposite. (7) apoptosis detection showed that the apoptosis rate of bone marrow cells in 200WLM radon exposure group and 400WLM group was significantly higher than that of control group, and the apoptosis rate of spleen and alveolar lavage cells was not significantly changed.
Conclusion: the decrease of thymus index, the change of the number of lymphocytes and neutrophils and the decrease of phagocytic function of macrophages after radon exposure showed that radon had toxic effects on immune cells and immune functions in rats, and the changes of reactive oxygen species, free calcium concentration, mitochondrial membrane potential and apoptosis rate after radon exposure were improved. It suggests that radon and its daughters may affect the immune function of the body through oxidative damage and cell cycle disorders.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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