实时细胞分析技术与Alamar Blue法用于测定纳米氧化铜细胞毒性的比较研究
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摘要
目的通过比较研究实时细胞分析技术(real time cell analyze,RTCA)及Alamar Blue法测定纳米氧化铜体外作用于人胚肺成纤维细胞MRC-5及人正常肺上皮细胞BEAS-2B的毒性作用,探讨RTCA实时细胞分析技术用于测定纳米材料体外细胞毒性的可行性。方法分别将MRC-5及BEAS-2B细胞接种于RTCA配套16孔板中及普通96孔板中,96孔板中细胞用于Alamar Blue法测定细胞毒性。分别用剂量为0.75g/L、0.38g/L、0.19g/L、0.094g/L、0.047g/L的纳米氧化铜混悬液进行染毒。选择12h、24h、36h、48h、60h时间点计算两种方法测得的IC50值。采用SPSS 16.0分析软件,利用配对t检验,对两种方法所得相同时间点的IC50值进行统计分析,判断其相关性及是否存在差异。结果 MRC-5细胞用RTCA法测定染毒后12 h、24 h、36 h、48 h、60 h的IC50值分别为391 mg/L、249 mg/L、185 mg/L、165mg/L、147mg/L;Alamar Blue法所得相同时间点的IC50值分别为507 mg/L、206 mg/L、172 mg/L、154mg/L、95.2mg/L。两种方法所测得IC50值进行统计分析,差异无统计学意义。BEAS-2B细胞用RTCA法测定染毒后12h、24h、36h、48h、60h的IC50值分别为131 mg/L、83.78 mg/L、65.37 mg/L、53.98mg/L、51.23 mg/L;Alamar Blue法所得相同时间点的IC50值分别为148 mg/L、104 mg/L、77.3mg/L、42.5mg/L、39.2mg/L。两种方法所测得IC50值进行统计分析,差异无统计学意义。结论RTCA实时监测法与Alamar Blue法在相同时间点测定纳米氧化铜体外细胞毒性得IC50值差异无统计学意义,说明RTCA实时监测法测定体外细胞毒性结果可靠,适用于纳米材料的体外毒性研究。
Objective To explore the applicability of RTCA real-time monitoring technology on the determination of cytotoxicity of nanometer materials by comparing the real time cell analyze(RTCA)method with Alamar Blue method. Methods MRC-5cells and BEAS-2Bcells were cultured in 16 well plate for RTCA analyzer and 96 well plate for Alamar Blue method for 27 hand then were challenged with nanometer copper-oxide suspension at different doses of 0.75g/L,0.38g/L,0.19g/L,0.094g/L and 0.047g/L,respectively.IC50 was calculated at 12 h,24h,36 h,48h,60 hafter the challenge for both methods.Paired t test by SPSS 16.0was used for data analysis. Results There were no significant differences of IC50 measured by RTCA compared with those by Alamar Blue method for MRC-5cells treated with nanometer copper-oxide for 12 h,24h,36 h,48hand 60h(391 vs 507mg/L;249 vs 206mg/L;185 vs 172mg/L;165 vs 154mg/L;147 vs 95.2mg/L),and for BEAS-2Bcells as well(131 vs 148 mg/L;83.78 vs 104 mg/L;65.37 vs 77.3 mg/L;53.98 vs 42.5mg/L;51.23 vs 39.2mg/L). Conclusions The differences of IC50 values acquired by RTCA real-time monitoring technology and Alamar Blue method at the same time points are not statistically significant,indicating that RTCA real-time monitoring technology can be used on the determination of cytotoxicity of nanometer material.
Objective To explore the applicability of RTCA real-time monitoring technology on the determination of cytotoxicity of nanometer materials by comparing the real time cell analyze(RTCA)method with Alamar Blue method. Methods MRC-5cells and BEAS-2Bcells were cultured in 16 well plate for RTCA analyzer and 96 well plate for Alamar Blue method for 27 hand then were challenged with nanometer copper-oxide suspension at different doses of 0.75g/L,0.38g/L,0.19g/L,0.094g/L and 0.047g/L,respectively.IC50 was calculated at 12 h,24h,36 h,48h,60 hafter the challenge for both methods.Paired t test by SPSS 16.0was used for data analysis. Results There were no significant differences of IC50 measured by RTCA compared with those by Alamar Blue method for MRC-5cells treated with nanometer copper-oxide for 12 h,24h,36 h,48hand 60h(391 vs 507mg/L;249 vs 206mg/L;185 vs 172mg/L;165 vs 154mg/L;147 vs 95.2mg/L),and for BEAS-2Bcells as well(131 vs 148 mg/L;83.78 vs 104 mg/L;65.37 vs 77.3 mg/L;53.98 vs 42.5mg/L;51.23 vs 39.2mg/L). Conclusions The differences of IC50 values acquired by RTCA real-time monitoring technology and Alamar Blue method at the same time points are not statistically significant,indicating that RTCA real-time monitoring technology can be used on the determination of cytotoxicity of nanometer material.
引文
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[2]Huang B,Li HJ,Jin DZ et al.Real-time cellular analysis for quantitative detection of functional Clostridium difficile toxin in stool[J].Expert Rev Molecu Diagnost,2014,14(3):281-291.
[3]JemaàM,Vitale I,Kepp O,et al.Selective killing of p53-deficient cancer cells by SP600125[J].EMBO Mol Med,2012,4(6):500-514.
[4]Garcia SN,Gutierrez L,McNulty A,et al.Real-time cellular analysis as a novel approach for in vitro cytotoxicity testing of medical device extracts[J].J Biomed Mater Res A,2013,101A(7):2097-2106.
[5]Ramis G,Martínez-Alarcón L,Quereda JJ,et al.Optimization of cytotoxicity assay by real-time,impedance-based cell analysis[J].Biomed Microdevices,2013,15(6):985-995.
[6]王天成,陈真,孟幻,等.纳米铜对小鼠毒性和血清常规生化指标影响的初步观察[J].卫生研究,2006,35(6):705-706.
[7]廖明阳,刘华钢.纳米铜的细胞毒性与溶出度分析[J].华西药学杂志,2011,26(1):41-44.
[8]孙婷婷,蒋澄宇.纳米氧化铜导致小鼠急性肺损伤[J].基础医学与临床,2012,32(4):386-389.
[9]孙婷婷.金属氧化物纳米材料的毒性研究[D].北京:协和医学院,2010.
[10]Teng Z,Kuang X,Wang J,et al.Real-time cell analysis-A new method for dynamic,quantitative measurement of infectious viruses and antiserum neutralizing activity[J].J Virol Methods,2013,193(2):364-370.