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(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 explore the immune cytotoxicity effect and its mechanism of trichloroethylene（ TCE） on activated human T cells. Methods a） Different concentrations of TCE（ 0. 32,0. 63,1. 25,2. 50,5. 00,10. 00 mmol / L）were used to treat activated T cells [activated with cluster of differentiation（ CD） 3 and CD28] respectively. Dimethyl sulfoxide（ DMSO） was used in the solvent group and the control group used no TCE or DMSO. The survival rate of activated T cells was calculated using CCK-8 assay after being cultured for 24 hours. b） Different concentrations of TCE（ 0. 00,2. 50,5. 00 mmol/L） were used to treat activated T cells. The apoptosis of cells was detected using flow cytometry. c） Different concentrations of TCE（ 0. 00,0. 32,0. 63,1. 25,2. 50,5. 00 mmol / L） were used to treat activated T cells and the level of cytokines as interleukin（ IL）-2 and IL-6 in cell culture supernatant was detected using enzyme linked immunosorbent assay after culturing for 24 hours. d） The control group and TCE treatment group of activated T cells were treated with 0. 00 and 5. 00 mmol / L TCE respectively. Cells were collected after culturing 0,30,60 and 120 minutes. Western Blot was used to detect the protein expression of signal transducers and activators of transcription3（ STAT3） and phospho-STAT3（ p-STAT3）. Results a） After 24-hour-exposure to TCE,the activated T cell survival rate of 10. 00 mmol / L TCE treatment group were significantly lower than that in the control group and DMSO group（ P〈0. 05）. b） There were no significant differences in cell apoptosis of activated T cells after treatment with 0. 00,2. 50 and5. 00 mmol / L TCE（ P〉0. 05）. c） In groups treated with different concentrations of TCE（ 0. 32,0. 63,1. 25,2. 50,5. 00 mmol / L）,the level of IL-2 and IL-6 in the cell culture supernatant of activated T cells were significantly higher than that in the control group（ P〈0. 05）. With the increasing of TCE exposure doses,the levels of IL-2 and IL-6 significantly increased（ P 0. 01） with dose-effect relationship. Compared with the control group,the levels of IL-17 A,interferongamma and transforming growth factor-beta in cell culture supernatant of activated T cells of the TCE treatment groups were no significant differences（ P〉0. 05）. d） The expression of p-STAT3 protein was low in the control group at different times. The expression of p-STAT3 protein in TCE treatment group was low at 0 minute,but increased at 30,60,120 minutes. The expression of p-STAT3 protein in TCE treatment group was higher than that in the control group at different time points. The levels of STAT3 total protein in TCE treatment group and the control group were similar at different time points,and were higher than the p-STAT3 proteins. Conclusion TCE at 5. 00 mmol / L had no observed toxic effect on activated T cells. High doses of TCE（ ≥10. 00 mmol / L） showed cytotoxic damages to activated T cells,and low doses of TCE（ ≤5. 00 mmol / L） could stimulate activated T cells to secrete IL-2 and IL-6. Treatment of TCE at 5. 00 mmol / L on activated T cells could up-regulated the level of p-STAT3.
出处
《中国职业医学》
CAS
北大核心
2017年第1期1-6,13,共7页
China Occupational Medicine
基金
国家科技支撑计划项目(2014BAI12B01)
国家自然科学基金(81673141)
国家临床重点专科建设项目(2011-09)
广东省职业病防治重点实验室(2012A061400007)
广东省科技计划项目(2014A020212551,2016A020215131)
广东省医学科研基金(A2014071,A2015624,B2015070)
