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目的研究三氯乙烯(TCE)对Jurkat T细胞免疫毒性,探讨TCE对Jurkat T细胞的最大无作用剂量。方法 (1)分别以不同浓度的TCE(0.10、0.50、1.00、2.00、5.00、10.00 mmol/L)作用于初始和活化Jurkat T细胞(以佛波酯和离子霉素进行活化处理),并设TCE浓度为0.00 mmol/L的对照组和二甲基亚砜(DMSO)组(溶剂对照组),在培养24、48、72 h后,光学显微镜下观察Jurkat T细胞形态,以CCK-8法检测Jurkat T细胞存活率。(2)分别采用不同剂量的TCE(0.00、0.02、0.20、2.00 mmol/L)作用于初始和活化Jurkat T细胞,分别在培养24、48、72 h后,以流式细胞术检测细胞凋亡情况,以酶联免疫吸附实验检测细胞培养上清中白细胞介素-2(IL-2)水平。结果 (1)染毒24 h后,10.00 mmol/L TCE染毒组可见明显细胞毒性,表现为细胞分散、体积变小、胞膜破裂、胞质浓缩、胞质内容物外溢逐渐增多。细胞存活率在染毒剂量与染毒时间的交互效应上有统计学意义(P<0.01),其中,在3个时间点,与同时间点对照组和DMSO组比较,0.10、0.50、1.00和2.00 mmol/L TCE染毒组细胞存活率均无出现有统计学意义的下降(P>0.05),5.00和10.00 mmol/L TCE染毒组细胞存活率均下降(P<0.01)。(2)以0.00~2.00mmol/L TCE染毒时,细胞凋亡率在染毒剂量主效应上以及其与细胞类型、染毒时间的交互效应上均无统计学意义(P>0.05)。活化Jurkat T细胞IL-2水平均高于同时间点同组初始Jurkat T细胞(P<0.01);在3个时间点,活化Jurkat T细胞IL-2水平均随TCE染毒剂量增加而增加,呈剂量-效应关系(P<0.01);活化Jurkat T细胞IL-2水平随TCE染毒时间增加而增加,呈时间-效应关系(P<0.01)。结论本研究条件下,TCE对Jurkat T细胞的最大无作用剂量为2.00 mmol/L。高剂量TCE(≥5.00 mmol/L)可对初始和活化Jurkat T细胞造成细胞毒性损伤;低剂量TCE(≤2.00 mmol/L)可刺激活化Jurkat T细胞IL-2分泌增高,并呈剂量-效应关系和时间-效应关系。
Objective To study the immunotoxicity of trichlorethylene (TCE) on Jurkat T cells and to explore the maximum non-effect dose of TCE on Jurkat T cells. Methods (1) TCE (0.10, 0.50, 1.00, 2.00, 5.00, 10.00 mmol / L) at different concentrations were applied to primary and activated Jurkat T cells (activated with phorbol ester and ionomycin) TCE concentration of 0.00 mmol / L and dimethylsulfoxide (DMSO) group (solvent control group), cultured for 24,48,72 h, under the light microscope Jurkat T cell morphology CCK-8 method Jurkat T cell survival was measured. (2) Tregs (0.00, 0.02, 0.20, 2.00 mmol / L) were administered to naive and activated Jurkat T cells at different doses, respectively. After 24, 48 and 72 hours of culture, apoptosis was detected by flow cytometry The levels of interleukin-2 (IL-2) in the cell culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). Results (1) After 24 h exposure, obvious cytotoxicity was observed in the 10.00 mmol / L TCE group, which was characterized by cell dispersion, smaller volume, ruptured membrane, concentration of cytoplasm and gradual increase of cytoplasmic contents. The cell survival rate was statistically significant (P <0.01) in terms of the interaction effect between the exposure dose and exposure time. Among the three time points, the cell survival rates were 0.10, 0.50, 1.00 and No significant difference was found in cell viability between 2.00 mmol / L TCE group and 5. 5 and 10.00 mmol / L TCE groups (P <0.01). (2) When treated with 0.00-2.00 mmol / L TCE, there was no significant difference in the rate of apoptosis between the two groups (P> 0.05) on the main effects of exposure dose and the interaction with cell types and exposure time. The levels of IL-2 in activated Jurkat T cells were higher than those of the same Jurkat T cells at the same time points (P <0.01). At 3 time points, the levels of IL-2 in activated Jurkat T cells increased with the doses of TCE, (P <0.01). The level of IL-2 in activated Jurkat T cells increased with the increase of TCE exposure time, showing a time-effect relationship (P <0.01). Conclusion Under the conditions of this study, the maximum non-effect dose of TCE on Jurkat T cells was 2.00 mmol / L. High-dose TCE (≥5.00 mmol / L) could induce cytotoxic damage on both primary and activated Jurkat T cells. Low-dose TCE (≤2.00 mmol / L) stimulated IL-2 secretion in activated Jurkat T cells with dose- Relationship and time - effect relationship.