内毒素对可吸入颗粒物所诱发的巨噬细胞毒性和前炎性细胞因子mRNA表达的影响

Role of Endotoxin in Cytotoxicity and Proinflammatory Cytokines mRNA Expression Induced by PM10 in RAW264.7 Cells

目的探讨可吸入颗粒物(PM10)所诱发的巨噬细胞毒性和前炎性细胞因子mRNA表达的改变与PM10中内毒素成分的关系。方法采用鲎试剂终点显色法测定未经处理的PM10(PM10+)和经130℃高热处理24 h的PM10(PM10-)中内毒素含量,将PM10+和PM10-以不同浓度(0、25、50、100、200、400μg/ml)分别染毒小鼠单核巨噬细胞系RAW264.7细胞24 h,采用MTT法测定细胞存活率,选取0、25、50、100、200μg/ml的PM10+和PM10-分别染毒RAW264.7细胞6、12、24 h,采用反转录-聚合酶链反应技术(RT-PCR)测定染毒各时相RAW264.7细胞白介素-6(IL-6)mRNA和肿瘤坏死因子-α(TNF-α)mRNA的表达水平。结果 PM10+和PM10-中内毒素含量分别为(58.7±6.4)EU/mg和(2.0±0.1)EU/mg。PM10+和PM10-对RAW264.7细胞的存活率影响不明显。染毒剂量在50μg/ml以上时,PM10+和PM10-均可使RAW264.7细胞IL-6 mRNA和TNF-αmRNA表达增加(P<0.05),随着染毒剂量的增加,IL-6 mRNA和TNF-αmRNA的表达均呈升高趋势,染毒6 h后,PM10-染毒组与PM10+染毒组相比,IL-6mRNA的表达减少(P<0.05)。结论 PM10对巨噬细胞的前炎性细胞因子mRNA表达的影响与其内毒素成分有关。

Objective To investigate the role of endotoxin in the cytotoxicity and proinflammatory cytokines mRNA expression induced by PM10. Methods Chromogenic end-point tachypleus amoebocyte lysate was used to quantify the endotoxin in PM10 （PM10^＋） and the heat-treated PM10 （PM10^-）. The mouse macrophage cell line RAW264.7 were exposed to PM10^＋ and PM10^- for 6 h, 12 h and 24 h at the concentrations of 0, 25, 50, 100, 200 μg/ml, respectively. The cytotoxicity was measured by MTT after exposure for 24 h. The expression of IL-6 mRNA and TNF-μ mRNA was measured by RT-PCR. Results The endotoxin concentration in PM10^＋ and PM10^- were （58.7±6.4） EU/mg and （2.0±0.1） EU/mg, respectively. After 24 h of treatment, neither PM10^＋ nor PM10^- showed obvious toxicity to RAW264.7 cells. Both PM10^＋ and PM10^- induced upregulated expression of IL-6 mRNA and TNF-α mRNA in dose-dependent manners （P〈0.05）. After 6 h of treatment, the expression of IL-6 mRNA induced by PM10^- were lower than that by PM10^＋（P〈0.05）. Conclusion The expression of proinflammatory cytokine mRNA induced by PM10 in RAW264.7 cell are correlated with its endotoxin content.