基于两种细胞的卷烟烟气体外免疫毒性评价

Evaluation of in vitro immunotoxicity of mainstream cigarette smoke by using two kinds of cells

为评价卷烟烟气总粒相物(TPM)的体外免疫毒性,选择小鼠淋巴瘤细胞(EL-4细胞)和小鼠巨噬细胞(Ana-1细胞)两种细胞,采用CCK-8法检测肯塔基3R4F参比卷烟烟气TPM染毒后细胞的存活率,采用Luminex液相芯片技术检测细胞上清液中20种细胞因子(FGF-basic、GM-CSF、IFN-γ、IL-1α、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、IL-13、IL-17、KC、IP-10、MCP-1、MIP-1α、VEGF、MIG、TNF-α)的分泌量。结果表明:两种细胞经烟气TPM染毒后,随着染毒剂量的增加,EL-4细胞中GM-CSF、IL-10和VEGF的分泌量均下调;Ana-1细胞中MCP-1、MIP-1α和TNF-α的分泌量均上调;两种细胞中其余17种细胞因子的分泌量无明显变化。不同类型/来源的细胞对烟气的毒性效应有不同的反应,因此采用两种细胞有助于更好地评价卷烟烟气的体外免疫毒性。该方法具有快速、灵敏、高通量等特点,适用于卷烟烟气体外免疫毒性评价。

To establish the in vitro immunotoxicity of cigarette smoke, mouse lymphoma cell EL-4 and mouse macrophage cell Ana-1 were treated with the total particular matter （TPM） in mainstream smoke of Kentucky 3R4F reference cigarette, then the viability of cells was measured by CCK-8 assay. Luminex liquid chip was used to determine the levels of 20 cytokines （FGF-basic, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, KC, IP-10, MCP-1, MIP-1α, VEGF, MIG, TNF-α） in the supernatant. The results showed that after exposure to TPM, GM-CSF, IL-10, VEGF were inhibited in EL-4, while MCP-1, MIP-1α, TNF-α were induced in Ana-1. The levels of the other 17 cytokines in two cell types did not change significantly with the increase of TPM concentration. The cell reactions of different types or sources to the toxicological effect of TPM of cigarette smoke differed, therefore adopting two kinds of cells is helpful to the better evaluation of in vitro immunotoxicity of cigarette smoke. The method is rapid, sensitive, high-throughput and suitable for assessing in vitro immunotoxicity of cigarette smoke.