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目的采用多种检测原理和观察终点的细胞毒性试验综合评估提取PM2.5的细胞毒性效应,为深入研究PM2.5生物学效应提供实验依据。方法用去离子水提取PM2.5,溶于二甲亚砜获得PM2.5悬液。不同浓度PM2.5染毒支气管上皮(HBE)细胞24 h,采用噻唑蓝比色(MTT)、乳酸脱氢酶释放法(LDH法)、台盼蓝拒染法、吖啶橙-溴化乙锭双染法(AO-EB双染法)4种细胞毒性实验评估PM2.5的细胞毒性,并计算半数抑制浓度(IC_(50))。结果去离子水提取的PM2.5溶于二甲亚砜后呈黑褐色,肉眼可见颗粒成分。不同浓度的PM2.5处理HBE细胞24 h后,台盼蓝拒染法(IC_(50)=815.5μg/m L)、LDH释放法(IC_(50)=827.3μg/m L)及AO-EB双染法(IC_(50)=887.5μg/m L)检测结果与空白对照组和溶剂对照组相比,细胞存活率均显著下降(P<0.05);而MTT法的细胞存活率检测结果显著高于对照组1.5~4.0倍(P<0.05),与细胞实际生长状态不符。结论用去离子水提取PM2.5可减少溶剂带入,能够最大程度反映空气污染实际情况;实验采用的4种方法都可检测出水提PM2.5细胞毒性,但由于样品颜色的特殊性,MTT实验不能准确检测本研究中PM2.5的细胞毒性。
Objective To evaluate the cytotoxicity effect of PM2.5 by comprehensive assessment of cytotoxicity test using multiple detection principles and observation endpoints, and provide experimental evidence for further study on the biological effects of PM2.5. Methods PM2.5 was extracted with deionized water and dissolved in dimethyl sulfoxide to obtain PM2.5 suspension. The cells were exposed to different concentrations of PM2.5 for 24 h. MTT assay, LDH assay, trypan blue exclusion assay, acridine orange-bromine bromide Four kinds of cytotoxicity experiments were used to evaluate the cytotoxicity of PM2.5, and the half inhibitory concentration (IC 50) was calculated. Results PM2.5 extracted with deionized water showed dark brown color when dissolved in dimethylsulfoxide and the granular composition was visible to the naked eye. After treated with different concentrations of PM2.5 for 24 h, the IC50 of 823.5 μg / ml and the IC50 of IC50 of 827.3 μg / ml and AO- EB double staining method (IC 50 = 887.5μg / m L), the cell viability was significantly decreased compared with the blank control group and the solvent control group (P <0.05), while the cell viability of the MTT assay Which was significantly higher than that in control group (P <0.05), which was not consistent with the actual cell growth. Conclusion PM2.5 can be extracted with deionized water to reduce the solvent introduction, which can reflect the actual situation of air pollution to the greatest extent. The four methods used in the experiment can detect the cytotoxicity of PM2.5 in water, but due to the particularity of sample color, MTT The experiment can not accurately detect the cytotoxicity of PM2.5 in this study.