RTCA与CCK-8法用于检测柴油废气颗粒物致支气管上皮细胞毒性的比较研究

Detection by RTCA and CCK-8 Assay to the Cytotoxicity of HBE Induced by Diesel Exhaust Particles

运用实时无标记细胞分析系统(RTCA)和Cell Counting Kit-8(CCK-8)法分别检测柴油废气颗粒物(DEP)致支气管上皮细胞(HBE)细胞毒性,从而对2种方法进行比较研究。分别以浓度为0、3.5、7、14、28和56 mg·L^(-1)2种柴油废气标准参考颗粒物(Standard Reference Material 1650b,SRM 1650b;Standard Reference Material 2975,SRM 2975)对HBE细胞进行暴露处理,分别暴露6、12、24和48 h后,检测不同DEP致HBE细胞毒性,比较各组之间细胞存活率或标准化细胞指数(normalized cell index,NCI)值的差异,考察2种方法的优缺点。并用细胞凋亡实验检测各差异组之间的凋亡率。在相同染毒浓度及暴露时间,与SRM 1650b相比,SRM 2975对HBE细胞的毒性更强。在RTCA检测DEP致HBE细胞毒性时,低浓度DEP组的NCI值已经表现出与对照组有统计学差异(P<0.05),而相同时间条件下,CCK-8法在更高浓度的DEP组才检测出显著的细胞活性下降(P<0.05)。且由细胞凋亡实验证实,与对照组相比,低浓度DEP组的细胞凋亡率已经有统计学差异。相对于CCK-8法,RTCA更适用于检测DEP致贴壁HBE细胞毒性。CCK-8法更适用于检测DEP致悬浮细胞的细胞毒性或与气液暴露装置联用时的贴壁/悬浮细胞毒性。

To compare the use of real-time cell analysis system(RTCA)and Cell Counting Kit-8(CCK-8)in detecting the toxic effects of diesel exhaust particles(DEP)on human bronchial epithelial(HBE)cells,the HBE cells were exposed to two types of Standard Reference Materials(SRM 1650b and SRM 2975)with the exposure concentrations of(0,3.5,7,14,28 and 56 mg·L^(-1)).After exposure for(6,12,24 and 48 h),the cytotoxicity of HBE cells induced by different DEP was detected.Then the advantages or shortcomings of RTCA and CCK-8 were analyzed by comparing the cell viability and normalized cell index(NCI)of different groups.And the cell apoptosis assay was used to detect the apoptosis rate between the different groups.Results show that:SRM 2975 was more toxic to HBE cells than SRM 1650b at the same exposure concentration and time.When RTCA was used to detect DEP-induced HBE cytotoxicity,the NCI value of low concentration DEP groups showed statistically significant difference(P<0.05).In comparison,the significant toxicity effects(P<0.05)were detected in relatively higher concentrations of DEP at the same exposure time by use of CCK-8 assay.And the cell apoptosis assay confirmed that the apoptosis rate of low concentration of DEP groups had already showed statistical difference compared with the control group(P<0.05).It is indicated that compared with CCK-8,RTCA was more suitable for detecting cytotoxicity of DEP-induced adherent HBE cells.And CCK-8 assays could be a better choice for detecting DEP-induced cytotoxicity of suspended cells or adhesion/suspension cytotoxicity when combined with air-liquid interface exposure systems.