【摘要】：目的：目前國(guó)內(nèi)外對(duì)氡及其子體的健康影響主要集中在其致癌作用,尤其是致肺癌作用的研究,而關(guān)于氡暴露對(duì)免疫系統(tǒng)毒性影響的研究較少。本課題通過(guò)觀察氡暴露對(duì)Wistar大鼠胸腺、脾臟組織及外周血、肺泡灌洗液以及骨髓細(xì)胞的損傷作用,探討氡對(duì)大鼠免疫功能的毒作用,為探索氡致機(jī)體免疫損傷的可能機(jī)制提供實(shí)驗(yàn)資料。 方法：選用健康雄性Wistar大鼠15只,體重100±10g,隨機(jī)分成三組,包括對(duì)照組、200工作水平月(WLM)組和400工作水平月組,每組5只。除對(duì)照組外,其余2組整體暴露于HD-3型多功能生態(tài)氡室,累積受照劑量分別達(dá)200和400WLM,劑量率保持恒定。大鼠麻醉后,腹主動(dòng)脈采血,收集支氣管肺泡灌洗液(BALF),胸腺、脾臟稱重后制成單細(xì)胞懸液,分離股骨取骨髓。計(jì)算胸腺、脾臟臟器指數(shù),CELL-DYN3700全自動(dòng)五分類(lèi)血液分析儀檢測(cè)BALF、骨髓、外周血細(xì)胞計(jì)數(shù)及分類(lèi),應(yīng)用流式細(xì)胞儀技術(shù)(FCM)和轉(zhuǎn)熒光質(zhì)粒細(xì)菌檢測(cè)肺泡巨噬細(xì)胞吞噬功能。取制備好的BALF、骨髓、外周血、胸腺和脾臟的單細(xì)胞懸液,通過(guò)相應(yīng)的熒光探針檢測(cè)不同劑量氡暴露組淋巴細(xì)胞及粒細(xì)胞內(nèi)活性氧(ROS)、游離鈣離子([Ca2+]i)濃度、線粒體膜電位(MMP)水平的變化；通過(guò)碘化丙啶(PI)染色觀察不同氡暴露組間脾臟、胸腺淋巴細(xì)胞凋亡率。 結(jié)果：(1)與對(duì)照組比較,200WLM氡暴露組胸腺指數(shù)明顯降低,而脾指數(shù)無(wú)明顯改變。大鼠外周血白細(xì)胞、中性粒細(xì)胞、淋巴細(xì)胞、嗜酸性粒細(xì)胞、嗜堿性粒細(xì)胞、紅細(xì)胞計(jì)數(shù)、血紅蛋白量、紅細(xì)胞壓積、平均血紅蛋白濃度及紅細(xì)胞分布寬度均明顯增加,而紅細(xì)胞平均體積明顯下降。400WLM氡暴露組紅細(xì)胞計(jì)數(shù)、血紅蛋白含量、紅細(xì)胞分布寬度明顯增加,而紅細(xì)胞平均體積、平均血紅蛋白量顯著降低。200WLM和400WLM氡暴露組單核細(xì)胞計(jì)數(shù)、血小板計(jì)數(shù)與對(duì)照組比較,均無(wú)顯著差異。氡暴露大鼠肺泡灌洗液淋巴細(xì)胞、中性粒細(xì)胞數(shù)量顯著增加,骨髓細(xì)胞總數(shù)、淋巴細(xì)胞及中性粒細(xì)胞計(jì)數(shù)均顯著增加。(2)200WLM氡暴露組巨噬細(xì)胞的吞噬率和吞噬指數(shù)顯著高于對(duì)照組,而400WLM氡暴露組肺泡巨噬細(xì)胞吞噬率無(wú)明顯改變,但吞噬指數(shù)顯著降低。(3)200WLM氡暴露組胸腺淋巴細(xì)胞ROS明顯增多,400WLM氡暴露組大鼠肺泡灌洗液粒細(xì)胞中的ROS較對(duì)照組顯著增加。(4)200WLM氡暴露組骨髓、外周血、肺泡灌洗液中淋巴細(xì)胞的線粒體膜電位(MMP)較對(duì)照組明顯降低,而400WLM組僅肺泡灌洗液中的淋巴細(xì)胞MMP顯著降低。(5)與對(duì)照組比較,200WLM組脾、胸腺淋巴細(xì)胞和肺泡灌洗液粒細(xì)胞鈣離子濃度明顯升高,而400WLM組肺泡灌洗液淋巴細(xì)胞的鈣離子濃度明顯升高,粒細(xì)胞鈣離子濃度則明顯下降。(6)細(xì)胞周期分析發(fā)現(xiàn),200WLM氡暴露組胸腺細(xì)胞G0/G1期細(xì)胞比例明顯降低,而S期細(xì)胞比例顯著升高,脾細(xì)胞則相反。(7)細(xì)胞凋亡檢測(cè)結(jié)果顯示,200WLM氡暴露組胸腺細(xì)胞及400WLM組骨髓細(xì)胞凋亡率明顯高于對(duì)照組,脾和肺泡灌洗細(xì)胞的凋亡率未見(jiàn)明顯改變。 結(jié)論：大鼠氡暴露后胸腺指數(shù)的減小,淋巴細(xì)胞和中性粒細(xì)胞數(shù)量的改變以及巨噬細(xì)胞吞噬功能的降低,表明氡對(duì)大鼠免疫細(xì)胞和免疫功能產(chǎn)生了毒性作用,而氡染毒后細(xì)胞內(nèi)活性氧、游離鈣離子濃度、線粒體膜電位和細(xì)胞凋亡率的改變,提示氡及其子體可能是通過(guò)氧化損傷和細(xì)胞周期障礙等途徑影響機(jī)體的免疫功能。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R114

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