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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(P<0.05)。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(P>0.05)。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(P<0.05);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(P<0.01),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(P>0.05)。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。
Objective To study the immunotoxicity of trichlorethylene (TCE) on human activated T cells and its mechanism. Method 1 Human T cells activated by differentiation antigen (CD3) and CD28 were treated with TCE at different doses (0.32, 0.63, 1.25, 2.50, 5.00 and 10.00 mmol / L) ) Group (solvent control group) and control group (without TCE and DMSO treatment). After cultured for 24 h, the survival rate of T cells was detected by CCK-8 colorimetric assay. 2 T cells activated by different doses of TCE (0.00, 2.50 and 5.00 mmol / L) for 24 h, the apoptosis of cells was detected by flow cytometry. 3 TAC cells were harvested and treated with TCE at different dosages (0.00,0.32,0.63,1.25,2.50 and 5.00 mmol / L) for 24 h. The levels of interleukin (IL) in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) ) -2 and IL-6 and other cytokines levels. 4 control group, the TCE cells exposed to TCE at dosage of 0.00 and 5.00 mmol / L, respectively, and TCE cells were harvested at 0, 30, 60 and 120 min after exposure. The signal transduction proteins and Transcriptional activator 3 (STAT3) and phosphorylated STAT3 (p-STAT3) protein expression. Results After 24 h exposure, the viability of T cells in 10 mmol / L TCE group was lower than that in control group and DMSO group (P <0.05). There was no significant difference in apoptotic rates between TCE treated with 2, 0.00, 2.50 and 5.00 mmol / L TCE (P> 0.05). The levels of IL-2 and IL-6 in supernatant of 3 T cells exposed to 0.32, 0.63, 1.25, 2.50 and 5.00 mmol / L TCE were higher than those of control (P <0.05) , The levels of IL-2 and IL-6 secreted by activated T cells increased (P <0.01), showing a dose-effect relationship. The expression levels of IL-17A, IFN-γ and TGF-β in the supernatant of the 5 TCE-exposed T cells were not significantly different from those in the control group (P> 0.05). The expression of p-STAT3 protein in activated T cells of control group was lower at each time point. The expression of p-STAT3 protein in activated T cells of TCE group was lower at 0 min, at 30, 60 and 120 min Up-regulated, and p-STAT3 protein expression at all time points were higher than the control group. The STAT3 total protein expression level of the two groups of activated T cells at each time point was relatively consistent, and both were relatively higher than the p-STAT3 protein. Conclusion The maximum effective dose of TCE to activated T cells is 5.00mmol / L. High dose of TCE (≥10.00 mmol / L) can cause cytotoxic damage on activated T cells; Low dose of TCE (≤5.00 mmol / L) can stimulate the secretion of IL-2 and IL-6 in activated T cells; L of TCE up-regulates STAT3 phosphorylation of activated T cells.