经饮水甲基汞对小鼠脾脏中成熟免疫细胞影响的时间-效应特征

Time-effect features of methyl mercury exposure via drinking on mature immune cells in spleen of mice

[目的]观察甲基汞暴露后不同时间点B10.S小鼠脾脏中髓系细胞、淋巴系细胞和骨髓中造血祖细胞生成相应成熟髓系细胞集落的数量及其动态变化,解释成熟免疫细胞动态变化的可能原因,探讨甲基汞引起机体免疫紊乱的可能机制。[方法]以6到8周龄的B10.S雌性小鼠为研究对象,随机分为对照组和实验组,分别饮用去离子水、1.25μmol/L甲基汞水溶液4周,测汞仪测定脾脏和脑中汞的含量,每周观察饮水和饮食消耗量。分别在第1周、2周和4周用动物电子称称量小鼠体重,并且在第1周、2周和4周用流式细胞仪检测小鼠脾脏中巨噬细胞、单核细胞、中性粒细胞、B淋巴细胞、CD4^+T细胞、CD8^+T细胞和自然杀伤(NK)细胞的数量,并用细胞集落形成试验(CFU,colony-formation units)检测骨髓造血祖细胞生成成熟髓系细胞的能力。[结果]2组小鼠饮水量、饮食消耗量、体重变化量之间的差异均无统计学意义(P>0.05)。饮用甲基汞溶液1周后,与对照组相比,小鼠脾脏中单核细胞、中性粒细胞在脾脏细胞中的百分比增高;第2周,单核细胞、巨噬细胞、中性粒细胞的数量百分比降低(P<0.05)。与对照组相比,B细胞数量百分比在第2周和第4周增高(P<0.05),CD4^+T细胞的数量百分比在第4周下降,CD8^+T细胞、NK细胞的数量百分比在第2周下降,差异均具有统计学意义(P<0.05)。CFU实验表明,在饮用甲基汞后第1周,粒细胞-红细胞-巨噬细胞-单核细胞集落(GEMM)数量、粒细胞-巨噬细胞集落(GM)数量、粒细胞集落(G)和巨噬细胞集落(M)数量增多,与对照组相比,差异有统计学意义(P<0.05);与对照组相比,饮用甲基汞2周后,GEMM和GM数量下降(P<0.05),4周后G和M数量增多(P<0.05)。[结论]甲基汞可导致小鼠骨髓中髓系祖细胞分化生成相应成熟细胞的能力先增强后减弱再恢复至正常水平,进而导致单核细胞、中性粒细胞、巨噬细胞的百分比呈现先升高后降低再逐渐恢复的趋势,CD4^+T细胞占脾脏细胞的百分比总体呈现下调的趋势,CD8^+T细胞、NK细胞占脾脏细胞的百分比先下调后上升,B细胞占脾脏细胞的比例总体呈现上调的趋势。甲基汞暴露后不同时间点小鼠脾脏中成熟免疫细胞的动态变化很复杂,总体上可以用CFU的变化来解释髓系细胞变化的原因。成熟免疫细胞的数量百分比随染毒时间的变化而发生改变,提示甲基汞引起的免疫紊乱可能与其导致的成熟免疫细胞比例失调有关。

[ Objective ] To explain the possible reasons of the dynamic changes of mature immune cells and explore the possible mechanism of immune disorder after methyl mercury （MeHg） exposure by observing the count and dynamic changes of splenic myeloid cells, lymphoid cells, and the generation of mature myeloid cells from hematopoietic progenitor cells in bone marrow at different time points following MeHg treatment.[ Methods ] Female B10.S mice at 6-8 weeks old were randomly divided into a control group and an experiment group, then were administrated with double distilled water or 1.25 μmol/L MeHg for 4 weeks. Mercury concentrations in spleen and brain were detected by mercury analyzer; drinking water and food consumption were observed weekly. After 1, 2, and 4 weeks of treatment, body weight was recorded with animal electronic scale; macrophages, monocytes, neutrophils, B lymphocytes, CD4＋T cells, CD8＋T cells, and nature killer （NK） ceils in spleen were detected by flow cytometry. Bone marrow cells were harvested through colony formation units （CFU） to assess the potential for CFU formation of functional progenitors in vitro.[ Results ] There was no significant difference between the two groups in drinking water and food consumption or body weight change （P〉 0.05）. The percentages of splenic monocytes and neutrophils at week 1 after drinking MeHg were higher than those of the control group, whereas the percentages of splenic monocytes, macrophages, and neutrophils were decreased at week 2 after exposure （P 〈 0.05）. The percentage of splenic B ceils was increased after 2 and 4 weeks of MeHg exposure （P 〈 0.05）, but the percentage of CD4＋T cells was decreased at week 4, and the percentage of CD8＋T cells and NK cells were decreased at week 2 （P 〈 0.05）. According to the CFU test, MeHg treatment increased the numbers of CFU for granulocyte-erythrocyte-monocyte-megakaryocyte （GEMM）, granulocyte-macrophage （GM）, granulocyte （G）, and macrophage （M） at week 1, and decreased the numbers of CFU for GEMM and GM at week 2, as compared with the control group （P〈 0.05）. After 4 weeks treatment, the numbers of CFU for G and M were increased compared with the control group （P 〈 0.05）.[ Conclusion ] The ability of bone marrow functional myeloid progenitors to differentiate into mature calls is enhanced, weakened, and then returned to the normal level after MeHg exposure. The percentages of monocytes, neutrophils, and macrophages show a similar pattern. At the same time, the percentage of splenic CD4＋T cells is down-regulated, the percentages of splenic CD8＋T cells and NK ceils decrease firstly and then increase, and the percentage of splenic B cells is up-regulated following MeHg exposure. The mature immune cells at different time points exhibit a complex dynamic change, which could be explained by the changes of CFU in general. The percentage of mature immune cells changes with the exposure time of MeHg, suggesting that the immune disorder induced by MeHg might be related to the disproportion of splenic mature immune ceils.