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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 investigate the toxic effects of nano-copper oxide on human embryo lung fibroblasts (MRC-5) and human normal human lung epithelial cells (BEAS-2B) in vitro through real-time cell analyze (RTCA) and Alamar Blue assay RTCA real-time cell analysis techniques for determining the feasibility of nanomaterials in vitro cytotoxicity. Methods MRC-5 and BEAS-2B cells were seeded into RTCA 16-well plates and normal 96-well plates respectively. The cells in 96-well plates were used for the cytotoxicity assay by Alamar Blue method. Respectively, with doses of 0.75g / L, 0.38g / L, 0.19g / L, 0.094g / L, 0.047g / L of nano-copper oxide suspension for exposure. IC50 values measured by the two methods were calculated at 12h, 24h, 36h, 48h and 60h. Using SPSS 16.0 analysis software, using the paired t-test, the IC50 values of the two methods at the same time point were statistically analyzed to determine the correlation and whether there are differences. Results The IC50 values of MRC-5 cells were 391 mg / L, 249 mg / L, 185 mg / L and 165 mg / L at 12 h, 24 h, 36 h, 48 h and 60 h after RTCA assay. 147mg / L. The IC50 values of Alamar Blue at the same time point were 507 mg / L, 206 mg / L, 172 mg / L, 154 mg / L and 95.2 mg / L, respectively. IC50 values measured by the two methods for statistical analysis, the difference was not statistically significant. The IC50 values of BEAS-2B cells were 131 mg / L, 83.78 mg / L, 65.37 mg / L, 53.98 mg / L and 51.23 mg / L at 12h, 24h, 36h, 48h and 60h respectively after RTCA assay. The IC50 values of Alamar Blue method at the same time point were 148 mg / L, 104 mg / L, 77.3 mg / L, 42.5 mg / L and 39.2 mg / L, respectively. IC50 values measured by the two methods for statistical analysis, the difference was not statistically significant. Conclusion There is no significant difference in the IC50 value between RTCA real-time monitoring method and Alamar Blue method in vitro cytotoxicity of nano-copper oxide, indicating that RTCA real-time monitoring of cytotoxicity in vitro results are reliable and suitable for in vitro toxicity of nano-materials.