烟草香料、电子烟与加热烟体外毒性评价研究

Study on inhalation toxicity of flavorings,e-cigarette and HTP in vitro

目的构建人支气管腺癌细胞模型(Calu-3),评估烟草香料、电子烟烟液和加热烟对呼吸道细胞毒性、炎症刺激反应以及细胞功能损伤的差异。方法采用CCK8细胞毒性分析方法检测不同烟草制品的细胞毒性,炎症基因的检测不同烟草制品诱导的炎症刺激反应,细胞生物学的方法检测Calu-3细胞特异性指标损伤:细胞跨上皮电阻、紧密连接、粘附连接等。结果不同剂量不同种类烟草制品可诱导细胞毒性增加,植物提取香料X02与X06,6.25 mg/ml剂量组诱导细胞增殖明显,细胞活性达到155.4%±4.43%和140.5%±4.25%(F=2745.81,P<0.01)。电子烟E02在1%剂量组可诱导细胞活性增加到118.2%±0.75%(F=639.03,P<0.01),高剂量时诱导细胞活性下降,由此激活了细胞毒物兴奋效应。低剂量的X01、X06、E02可诱导促炎症因子IL-1β、IL-6、IL-8、IL-18、TNF-α和TGF-β基因的相对表达量上调,粘液基因MUC5AC的相对表达量下降,紧密连接蛋白ZO-1与粘附连接蛋白E-Cadherin的蛋白表达发生改变。结论气道上皮细胞在烟草制品低剂量暴露时,可诱导细胞结构与功能受损,从而影响呼吸道上皮细胞正常的粘液清除与防御机制。

Objective To study the characteristics of a human bronchial epithelial cell line(Calu-3)and to evaluate the differenct effects of tobacco flavoring chemicals,e-cigarette liquid and heated tobacco products(HTP)on respiratory cytotoxicity,inflammatory response and cell function damage.Methods CCK8 cytotoxicity analysis method was used to detect the cytotoxicity of different tobacco products,inflammatory gene was used to detect the inflammatory response induced by tobacco products,and the specific physiological indicators of Calu-3 cells,such as transepithelial electrical resistance(TEER),tight junction(TJ),adhesion junction(AJ)and other cell functions are detected.Results Different doses of tobacco products could induce increased cytotoxicity.Flavoring X02,X06 and e-cigarette E02 could induce cell proliferation at low dose,the cell viability of 6.25 mg/ml group increased up to 155.4%±4.43%,140.5%±4.25%(F=2745.81,P<0.01)and 1%group increased up to 118.2%±0.75%(F=639.03,P<0.01).Conversely,Flavorings induced cell viability were decreased at high dose.Low dose groups X01,X06,E02 induced up-regulation of pro-inflammatory cytokines IL-1β,IL-6,IL-8,IL-18,TNF-αand TGF-βand they decreased the expression of MUC5AC.The characteristics of tight junction protein ZO-1 and adhesion junction protein E-cadherin were changed.Conclusion The structure and function of airway epithelial cells may be impaired when exposed to tobacco products at low dose,which may affect the normal mucus clearance and defense mechanism of airway epithelial cells.