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目的探讨交通相关的细颗粒物(PM2.5)对人外周血淋巴细胞的免疫毒性,从钙信号途径揭示免疫抑制机制。方法采用0、5、20、80、320、800μg/ml PM2.5染毒T淋巴细胞24、48 h,采用四甲基偶氮唑盐(MTT)比色法测定T淋巴细胞的增殖功能。采用0、50、100、200μg/ml PM2.5染毒T淋巴细胞244、8 h,采用流式细胞仪测定T淋巴细胞的免疫功能(CD3+/CD4+/CD8+亚群和CD4+/CD8+)。采用荧光分光光度计测定T淋巴细胞的钙离子浓度([Ca2+]i)。用试剂盒测定T淋巴细胞的Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活力。结果 320、800μg/ml PM2.5可抑制细胞增殖功能,且与对照组比较有统计学意义(P<0.05);50、200μg/ml PM2.5染毒细胞24 h,染毒组T淋巴细胞亚群(CD3+、CD4+百分含量、CD4+/CD8+比值)与对照组比较变化不明显;50、100、200μg/ml PM2.5染毒细胞48 h后,染毒组T淋巴细胞亚群(CD3+、CD4+百分含量、CD4+/CD8+比值)均低于对照组,且各剂量组CD3+和200μg/ml CD4+百分含量与对照组比较,差异有统计学意义(P<0.05)。200μg/mlPM2.5染毒细胞1、3、61、2、24 h后[,Ca2+]i均高于对照组,除24 h外,与对照比较,差异均具有统计学意义(P<0.05)。在3 h时胞内[Ca2+]i最高,随时间延长[,Ca2+]i逐渐降低。100、200μg/ml PM2.5染毒细胞3 h时,[Ca2+]i高于对照组,差异有统计学意义(P<0.05);随染毒浓度的升高,Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活力有降低趋势,且Na+-K+-ATP酶活力在200μg/mlPM2.5组与对照组比较,差异有统计学意义(P<0.05)。结论交通相关的PM2.5可引起人外周血淋巴细胞免疫抑制,其可能与钙稳态失衡有关。
Objective To investigate the immunotoxicity of traffic-related fine particles (PM2.5) to human peripheral blood lymphocytes and to reveal the immunosuppressive mechanism from calcium signaling. Methods T lymphocytes were exposed to PM2.5 at 0, 5, 20, 80, 320, 800μg / ml for 24,48 h. The proliferation of T lymphocytes was measured by MTT assay. T lymphocytes were exposed to PM2.5 at 0, 50, 100, 200μg / ml for 244,8 h. The immune function (CD3 + / CD4 + / CD8 + subsets and CD4 + / CD8 +) of T lymphocytes were measured by flow cytometry. The T-lymphocyte calcium concentration ([Ca 2+] i) was measured using a fluorescence spectrophotometer. The activity of Na + -K + -ATPase and Ca2 + -Mg2 + -ATPase in T lymphocytes were measured by kit. Results PM2.5 at 320 and 800μg / ml could inhibit cell proliferation and had statistical significance compared with the control group (P <0.05). Twenty-four hours after exposure to 50 and 200μg / ml PM2.5, T lymphocytes (CD3 +, CD4 + percentage, CD4 + / CD8 + ratio) had no obvious change compared with the control group. After treated with 50, 100 and 200μg / ml PM2.5 for 48 h, the T lymphocyte subsets (CD3 + , CD4 + percentage, CD4 + / CD8 + ratio) were lower than that of the control group, and the percentage of CD3 + and 200μg / ml CD4 + in each dose group was significantly higher than that in the control group (P <0.05). After exposure to 200μg / ml PM2.5 for 1, 3, 61, 2 and 24 hours, the expression of [, Ca2 +] i were higher in the control group than in the control group. The differences were statistically significant (P <0.05) . The intracellular [Ca2 +] i was the highest at 3 h and gradually decreased with time [Ca2 +] i. Compared with the control group, the expression of [Ca2 +] i was significantly increased at 100 and 200μg / ml PM2.5 exposure for 3 h (P <0.05). With the increase of the concentration of Na + -K + -ATPase and The activity of Ca2 + -Mg2 + -ATP decreased, and the activity of Na + -K + -ATPase in 200μg / ml PM2.5 group compared with the control group, the difference was statistically significant (P <0.05). Conclusion Traffic-related PM2.5 can cause immunosuppression in human peripheral blood lymphocytes, which may be related to the imbalance of calcium homeostasis.