四种细胞毒性试验测定柴油机尾气颗粒物提取物急性毒性的比较  

Comparison of four cytotoxicity assays for determining the acute toxicity of Diesel Exhaust Particulate extracts

作　　者：李雪[1] 牛勇[2] 贾强[2] 张哲[2] 段化伟[2] 王学生[1] 郑玉新[2] 

机构地区：[1]河北联合大学公共卫生学院,唐山063000 [2]中国疾病预防控制中心职业卫生与中毒控制所化学安全与健康重点实验室

出　　处：《卫生研究》2013年第2期221-227,共7页Journal of Hygiene Research

基　　金：国家自然科学基金重点项目(No.81130050);卫生行业科研专项基金(No.200902006)

摘　　要：目的比较四种体外细胞毒性试验检测柴油机尾气颗粒物(DEP)提取物急性毒性的能力。方法用浓度分别为5、10、20、40、80和160μg/ml的DEP提取物作用于16HBE细胞,于12、24及48h后,通过MTT法、中性红法、乳酸脱氢酶(LDH)法及蛋白测定法测细胞存活率。结果 MTT法在检测急性毒性方面与其它三种方法相比,染毒12h时就表现出了很高的敏感性。随着染毒时间的增加,MTT法与中性红法的敏感性逐渐提高,染毒24h后与对照组相比,各剂量组细胞存活率明显降低(P<0.05)。LDH法测定DEP提取物染毒24h后细胞存活率有所降低,但继续增加染毒时间,存活率不再发生变化。蛋白法测定染毒12h和24h的细胞存活率,仅高剂量组显著降低,染毒48h后,各剂量组细胞存活率均显著降低。结论四种不同的细胞毒性试验测定结果存在差异,MTT法和中性红法比蛋白法和LDH法更为敏感。结合各试验检测的毒性指标可以看出,DEP提取物主要影响16HBE细胞内各细胞器的功能,如线粒体、溶酶体;对于细胞贴壁的影响和细胞膜损伤程度的影响较弱。Objective To compare the determination ability of fom in vitro cytotoxicity assays for acute toxicity of diesel exhaust particulate （DEP） extracts. Method The cell viability of cultured 16HBE cells was measured by the MTT assay, the neutral red assay, the lactate dehydrogenase （LDH） leakage assay and the determination of total protein content after 12, 24 and 48h of being exposed to DEP extracts at different concentrations of 5,10,20,40,80 and 160p^g/ml. Results For measuring the acute cytotoxicity, the MTT assay was more delicate than the other three methods after 12h exposure. The sensitivity of both the MTT assay and the neutral red assay increased as the exposure time extended. The cell viability at each dosage decreased significantly after being exposed to DEP extracts for 24h compared to the control group （P 〈 0.05）. When using the LDH assay, the cell viability reduced after 24h exposure to DEP extracts, but changed no more as time prolonged. Only the cell viabilities of the groups exposed to the high dosages of DEP extracts were observed with significant reduction by the protein assay after 12h and 24h exposure respectively. The cell viabilities measured by the same method at all dosages decreased after 48h exposure. Conclusion The results of the four eytotoxieity assays showed differences in the present study. The MTT assay and the neutral red assay are more sensitive in detecting the adverse effect to 16HBE cells done by DEP extracts compared to the LDH assay and the protein assay. Taken the indicants of each toxicity assay into account, the DEP extracts may have major adverse effects on the functions of the organelles in 16HBE cells, such as mitoehondria and lysosomes, however little influence to the cell anchorage dependence and the cell membrane damage.

关 键 词：柴油机尾气颗粒物提取物 MTT 中性红 乳酸脱氢酶 细胞毒性 

分 类 号：X513[环境科学与工程—环境工程] R994.6[医药卫生—毒理学]