【作者】 王爱清；

【导师】 童建；

【作者基本信息】 苏州大学 ， 卫生毒理学， 2013， 硕士

【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。更多还原

【Abstract】 Objective:Recent studies on the health effects of radon and its daughters are mainly focused in its carcinogenicity, especially lung cancer, while its toxic effects on immune system are rare. This study is to explore the adverse effects of radon and its daughters on immune functions of thymus, spleen, and cells of peripheral blood and bronchoalveolar lavage fluid, to provide experimental data for mechanistic investigation of radon-induced immune damage in rats.Methods:Fifteen male Wistar rats weighted100±10g were randomly divided into three groups, i.e., control group,200WLM and400WLM (n=5)group.Rats were exposed to HD-3multifunction ecological radon chamber at the cumulative radiation doses of200and400working level months(WLM). At the end of the exposure, the spleen and thymus were aseptically excised and weighed, and bronchoalveolar lavage fluid(BALF)、peripheral blood、bone marrow(BM)were collected to obtain single cells of the spleen and thymus. The total number and proportion of cells in BALF、BM and in the peripheral blood were counted. The phagocytosis of alveolar macrophage was detected by flow cytometry (FCM) using bacterial which transferred the fluorescent plasmid. The level of reactive oxygen species (ROS), intracellular free calcium concentration ([Ca2+]i), mitochondrial membrane potential (MMP), cell cycle and the apoptosis in spleen, bone marrow, BALF and thymic lymphocytes were also detected by FCM.Results:(1). Compared with the control group, the thymus index of200WLM group was significantly lower. The peripheral blood leukocytes, neutrophils, lymphocytes, eosinophils, basophils, red blood cell, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, and red blood cell distribution width increased obviously in200WLM group. in In the400WLM group, hemoglobin, red blood cell count and distribution width dramatically increased and the mean corpuscular volume, mean corpuscular hemoglobin (MCH) significantly decreased. The number of lymphocytes and neutrophils in BALF, as well as the total number of bone marrow cells, lymphocytes and neutrophil also increased.(2). The alveolar macrophage phagocytosis of200WLM group was significantly higher than the control group while the phagocytic index of alveolar macrophage in400WLM group was significantly reduced.(3). Compared with the control, the level of ROS of thymus lymphocytes in200WLM group and BALF granulocyte in400WLM group was significantly higher than in the control group.(4) The MMP of lymphocytes in bone marrow, peripheral blood and BALF in200WLM group, and BALF lymphocytes in400WLM group was significantly lower.(5) Compared with the control group, the intracellular Ca2+concentration of lymphocytes of spleen, thymus and BALF granulocytes were significantly higher in200WLM group. In400WLM group, The Ca2+concentration of lymphocytes of BALF increased and that of BALF granulocytes decreased.(6) The G0/G1phase of thymocytes was reduced and the S phase was prolonged in200WLM group. The opposite change was observed in400WLM group.(7) The apoptotic rate of thymocytes and bone marrow cells in both radon exposed groups was significantly higher than the control group.Conclusion:The adverse effects of radon exposure in terms of induce of the thymus index, changes in number of lymphocytes and neutrophils in bone marrow and bronchoalveolar lavage fluid, and impaired ability of macrophages, demonstrated a toxic action of radon on the immune system. Changes in ROS production, intracellular Ca2+concentration, level of MMP and apoptosis all suggested that the adverse immunological effect of radon was mediated by ways of oxidative damage and cell cycle blockage.更多还原