三氯乙烯对人活化T细胞免疫毒性作用及机制研究

发布时间：2018-04-27 04:10

  本文选题：三氯乙烯 + T细胞　； 参考：《中国职业医学》2017年01期

【摘要】：目的研究三氯乙烯(TCE)对人活化T细胞的免疫毒性及其作用机制。方法 1取经分化抗原(CD3)和CD28活化的人T细胞,分别予不同剂量(0.32、0.63、1.25、2.50、5.00、10.00 mmol/L)的TCE染毒,并设二甲基亚砜(DMSO)组(溶剂对照组)和对照组(不予TCE和DMSO处理);培养24 h后,以CCK-8比色法检测T细胞的存活率。2以不同剂量TCE(0.00、2.50和5.00 mmol/L)染毒活化T细胞24 h后,以流式细胞术检测细胞凋亡情况。3取活化T细胞,分别予不同剂量(0.00、0.32、0.63、1.25、2.50、5.00 mmol/L)的TCE染毒24 h后,以酶联免疫吸附实验检测培养上清中白细胞介素(IL)-2和IL-6等细胞因子水平。4对照组、TCE染毒组活化T细胞分别予剂量为0.00和5.00 mmol/L的TCE染毒,于染毒0、30、60和120 min时收获细胞,以免疫印迹法检测信号传导蛋白和转录激活物3(STAT3)及磷酸化STAT3(p-STAT3)的蛋白表达情况。结果 1染毒24 h后,10.00 mmol/L TCE染毒组活化T细胞的存活率分别低于对照组和DMSO组(P0.05)。2 0.00、2.50和5.00 mmol/L剂量的TCE作用于活化T细胞时,细胞凋亡率差异无统计学意义(P0.05)。3 0.32、0.63、1.25、2.50和5.00 mmol/L TCE染毒组活化T细胞培养上清液中IL-2与IL-6水平均高于对照组(P0.05);随着TCE染毒剂量的增加,活化T细胞分泌的IL-2和IL-6水平均上升(P0.01),均呈剂量-效应关系。5个TCE染毒组活化T细胞培养上清液中IL-17A、IFN-γ和TGF-β表达水平分别与对照组比较,差异均无统计学意义(P0.05)。4对照组活化T细胞在各个时间点p-STAT3蛋白的表达均较低;TCE染毒组活化T细胞p-STAT3蛋白表达在0 min时间点较低,在30、60和120 min时间点均上调,且各个时间点p-STAT3蛋白表达均高于对照组。2组活化T细胞在各个时间点的STAT3总蛋白表达水平较为一致,且均相对高于p-STAT3蛋白。结论 TCE对活化T细胞的最大无作用剂量为5.00mmol/L。高剂量TCE(≥10.00 mmol/L)可对活化T细胞造成细胞毒性损伤;低剂量TCE(≤5.00 mmol/L)可刺激活化T细胞IL-2和IL-6分泌增加;浓度为5.00 mmol/L的TCE可上调活化T细胞的STAT3磷酸化水平。
[Abstract]:Objective to study the immunotoxicity of trichloroethylene (TCE) on human activated T cells and its mechanism. Methods 1 Human T cells activated by differentiation antigen (CD3) and CD28 were treated with different doses of TCE of 0.32 ~ 0.63 ~ 1.25 ~ 2.50 ~ 5.00 mmol 路L ~ (-1) and treated with dimethyl sulfoxide (DMSO) group (solvent control group) and control group (TCE and DMSO), respectively, and cultured for 24 h, and then cultured for 24 h, then treated with DMSO (dimethyl sulfoxide) group (solvent control group) and control group (TCE and DMSO). The survival rate of T cells was detected by CCK-8 colorimetry. 2. The survival rate of T cells was measured by CCK-8 colorimetry. The T cells were activated with different doses of TCEC 0.002.50 and 5.00 mmol / L for 24 h. The apoptosis of T cells was detected by flow cytometry. The activated T cells were obtained by flow cytometry. The activated T cells were treated with TCE of 0.000.32 ~ 0.32 ~ (0.32) ~ 1.252.50 mmol / L for 24 h, respectively. Enzyme linked immunosorbent assay (Elisa) was used to detect the levels of cytokines such as interleukin-2 and IL-6 in the supernatant. The activated T cells in the control group were treated with TCE at doses of 0.00 and 5.00 mmol/L, respectively, and the cells were harvested at 30 and 120 min after exposure. The protein expression of signal transduction protein, transcriptional activator 3 (STAT3) and phosphorylated STAT3 (p-STAT3) were detected by Western blotting. Results (1) the survival rate of activated T cells in the 10.00 mmol/L TCE group was lower than that in the control group and DMSO group after 24 hours of exposure. The survival rate of the activated T cells was significantly lower than that of the control group and the DMSO group. The levels of IL-2 and IL-6 in the supernatant of activated T cell culture in the groups exposed to mmol/L TCE were higher than those in the control group, and the levels of IL-2 and IL-6 in the supernatant of activated T cell culture were higher than those in the control group, and with the increase of the dose of TCE, there was no significant difference in apoptosis rate between the two groups. The levels of IL-2 and IL-6 secreted by activated T cells increased in a dose-effect relationship, and the expression levels of IL-17An- 纬 and TGF- 尾 in the supernatants of activated T cells in the five TCE exposed groups were compared with those in the control group, respectively. The expression of p-STAT3 protein in activated T cells of control group was lower at 0 min time point than that in control group at all time points, and increased at 30 ~ 60 and 120 min time points. The expression of p-STAT3 protein at each time point was higher than that of the control group. 2. The expression level of total STAT3 protein in activated T cells at each time point was consistent, and the expression level of p-STAT3 protein was higher than that of p-STAT3 protein. Conclusion the maximum nonactive dose of TCE on activated T cells is 5.00 mmol / L. High dose TCE (鈮，

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