2种方法测定氧化铜纳米颗粒对新生大鼠脑微血管内皮细胞毒性的比较研究
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摘要
目的:比较实时细胞分析技术(RTCA)和刃天青法测定氧化铜纳米颗粒(CuO-NPs)对新生大鼠脑微血管内皮细胞(BMECs)的细胞毒性的异同。方法:采用RTCA法确定BMECs细胞毒性实验的最佳细胞接种数量及染毒时间。分别用1.5、0.75、0.38、0.19、0.09 mg/mL的CuO-NPs染毒BMECs,以无细胞的培养液为对照组(0 mg/mL),分别以RTCA法及刃天青法比较其细胞毒性。通过比较2种方法所获得的IC_(50)值判断2种毒性测试方法的相关性。结果:每孔2.5×10~3个细胞为BMECs最佳接种浓度,接种后40h为最佳染毒时间。RTCA检测结果表明1.5、0.75 mg/mL的CuO-NPs染毒BMECs后约2 h开始细胞活性降低,并在染毒4 h内全部死亡。通过刃天青法分别在染毒3、12、24、36、48 h进行测定,得到与RTCA相似的结果,即1.5和0.75 mg/mL组的细胞毒性最大,且CuO-NPs对BMECs的细胞毒性呈剂量依赖性趋势。相关性分析结果显示2种方法所获得IC_(50)值相关系数为0.999,P=0.000,具相关性。配对t检验结果显示,差异无统计学意义(t=1.125,P=0.323)。从计算IC_(50)的R~2值来看,RTCA拟合曲线法R~2值高于刃天青法R~2值。结论:CuO-NPs体外作用于BMECs具有剂量依赖性的细胞毒性,RTCA方法与刃天青法测定BMECs细胞毒性结果具有相关性,RTCA方法所得结果可能更为准确,更适合于CuO-NPs的细胞毒性研究。
OBJECTIVES: Use of two methods to compare the cytotoxicity of CuO nanoparticles on brain microvascular endothelial cells(BMECs) in neonatal rats. METHODS: Growth curves of BMECs were determined by Real-Time Cell Analysis(RTCA) which was used to determine the optimal number of cells to be inoculated and the optimal exposure time for the toxicity experiment. Cells were treated with CuO-NPs at 1.5, 0.75, 0.38, 0.19, 0.09 mg/mL, and the culture medium without cell was used as control(0 mg/mL). The cytotoxicity of CuO-NPs was analyzed by two methods: RTCA and Resazurin. Correlations of the two methods were determined by comparing the IC_(50) values obtained. RESULTS: The growth curves of BMECs show that 2.5 × 10~3 cells/well was the optimal inoculation concentration, and 40 h after inoculation was the best exposure time. RTCA results show that BMECs were transiently unstable at 2-3 h after exposure to 1.5, 0.75, and 0.38 mg/mL CuO-NPs, followed by progressive death. The cell viability of BMECs at the 1.5 and 0.75 mg/mL groups began to decrease at about 2 h after exposure, and all cells died at 4 h. The cell viability was measured by the Resazurin method at 3,12,24,36,and 48 h after exposure from the same doses. The Resazurin method yielded similar results to RTCA, ie,the 1.5 and 0.75 mg/mL groups had the greatest cytotoxicity, and the cytotoxicity of CuO-NPs on BMECs showed a dose-dependent trend. Results from correlation analyses show that IC_(50) obtained by the two methods has a good correlation, and correlation coefficient was 0.999, P=0.000. Paired t test results show the difference was not statistically significant(t=1.125,P=0.323). From the calculation of R~2 value of IC_(50), R~2 value of RTCA fitting curve is higher than R~2 value of the Resazurin method Logistic regression coefficient. CONCLUSION : CuO-NPs demonstrated dosedependent cytotoxic effects on BMECs in vitro. The results of cytotoxicity of BMECs as determined by the RTCA and Resazurin assays were related, but results obtained by the RTCA method appear to be more accurate. Therefore,RTCA may be more suitable for the research of cytotoxicity of CuO-NPs.
OBJECTIVES: Use of two methods to compare the cytotoxicity of CuO nanoparticles on brain microvascular endothelial cells(BMECs) in neonatal rats. METHODS: Growth curves of BMECs were determined by Real-Time Cell Analysis(RTCA) which was used to determine the optimal number of cells to be inoculated and the optimal exposure time for the toxicity experiment. Cells were treated with CuO-NPs at 1.5, 0.75, 0.38, 0.19, 0.09 mg/mL, and the culture medium without cell was used as control(0 mg/mL). The cytotoxicity of CuO-NPs was analyzed by two methods: RTCA and Resazurin. Correlations of the two methods were determined by comparing the IC_(50) values obtained. RESULTS: The growth curves of BMECs show that 2.5 × 10~3 cells/well was the optimal inoculation concentration, and 40 h after inoculation was the best exposure time. RTCA results show that BMECs were transiently unstable at 2-3 h after exposure to 1.5, 0.75, and 0.38 mg/mL CuO-NPs, followed by progressive death. The cell viability of BMECs at the 1.5 and 0.75 mg/mL groups began to decrease at about 2 h after exposure, and all cells died at 4 h. The cell viability was measured by the Resazurin method at 3,12,24,36,and 48 h after exposure from the same doses. The Resazurin method yielded similar results to RTCA, ie,the 1.5 and 0.75 mg/mL groups had the greatest cytotoxicity, and the cytotoxicity of CuO-NPs on BMECs showed a dose-dependent trend. Results from correlation analyses show that IC_(50) obtained by the two methods has a good correlation, and correlation coefficient was 0.999, P=0.000. Paired t test results show the difference was not statistically significant(t=1.125,P=0.323). From the calculation of R~2 value of IC_(50), R~2 value of RTCA fitting curve is higher than R~2 value of the Resazurin method Logistic regression coefficient. CONCLUSION : CuO-NPs demonstrated dosedependent cytotoxic effects on BMECs in vitro. The results of cytotoxicity of BMECs as determined by the RTCA and Resazurin assays were related, but results obtained by the RTCA method appear to be more accurate. Therefore,RTCA may be more suitable for the research of cytotoxicity of CuO-NPs.
引文
[1]陈春英,高愈希.纳米材料的环境、生物行为及效应研究专辑:序言[J].生态毒理学报,2016,(5):1.
[2]马艺嘉.纳米材料暴露与应对[J].现代职业安全,2016,(1):92-93.
[3]段链,顾雯,王秦,等.实时动态监测纳米氧化铜作用于CHL细胞的毒性效应[J].癌变·畸变·突变,2016,28(3):205-208.
[4]赵康峰,候智全,张丽霞,等.纳米氧化铜对豚鼠血脑屏障通透性影响观察[J].毒理学杂志,2017,(5):340-344.
[5]HU Y L,GAO J Q.Potential neurotoxicity of nanoparticles[J].Int J Pharm,2010,394(1):115-121.
[6]STOLERMAN L P,LAWRENCE H.Neurovascular Unit[M].Heidelberg:Springer Berlin Heidelberg,2015:1099-1099.
[7]KRIZBAI I,FAZAKAS C,NAGYOSZI P,et al.The role of the blood-brain barrier in the formation of brain metastases[J].Int J Mol Sci,2013,14(1):1383-1411.
[8]COMBES V,GUILLEMIN G J,CHAN-LING T,et al.The crossroads of neuroinflammation in infectious diseases:endothelial cells and astrocytes[J].Trends Parasitol,2012,28(8):311-319.
[9]O'BRIEN J,ORTON T P F,WILSON I.Investigation of the Alamar Blue(resazurin)fluorescent dye for the assessment of mammalian cell cytotoxicity[J].Eur JBiochem,2000,267:5421-5426.
[10]DI C E,INGANGI V,ANGELINI C,et al.Amacroscopic mathematical model for cell migration assays using a real-time cell analysis[J].PLoS ONE,2016,11(9):e0162553.
[11]BIHARI P,VIPPOLA M,SCHULTES S,et al.Optimized dispersion of nanoparticles for biological in vitro and in vivo studies[J].Part Fibre Toxicol,2008,5(1):14-14.
[12]MOE B,GABOS S,LI X F.Real-time cell-microelectronic sensing of nanoparticle-induced cytotoxic effects[J].Anal Chim Acta,2013,789(14):83-90.
[13]张智勇.纳米毒理学研究方法与实验技术[M].北京:科学技术出版社,2015:157-177.
[2]马艺嘉.纳米材料暴露与应对[J].现代职业安全,2016,(1):92-93.
[3]段链,顾雯,王秦,等.实时动态监测纳米氧化铜作用于CHL细胞的毒性效应[J].癌变·畸变·突变,2016,28(3):205-208.
[4]赵康峰,候智全,张丽霞,等.纳米氧化铜对豚鼠血脑屏障通透性影响观察[J].毒理学杂志,2017,(5):340-344.
[5]HU Y L,GAO J Q.Potential neurotoxicity of nanoparticles[J].Int J Pharm,2010,394(1):115-121.
[6]STOLERMAN L P,LAWRENCE H.Neurovascular Unit[M].Heidelberg:Springer Berlin Heidelberg,2015:1099-1099.
[7]KRIZBAI I,FAZAKAS C,NAGYOSZI P,et al.The role of the blood-brain barrier in the formation of brain metastases[J].Int J Mol Sci,2013,14(1):1383-1411.
[8]COMBES V,GUILLEMIN G J,CHAN-LING T,et al.The crossroads of neuroinflammation in infectious diseases:endothelial cells and astrocytes[J].Trends Parasitol,2012,28(8):311-319.
[9]O'BRIEN J,ORTON T P F,WILSON I.Investigation of the Alamar Blue(resazurin)fluorescent dye for the assessment of mammalian cell cytotoxicity[J].Eur JBiochem,2000,267:5421-5426.
[10]DI C E,INGANGI V,ANGELINI C,et al.Amacroscopic mathematical model for cell migration assays using a real-time cell analysis[J].PLoS ONE,2016,11(9):e0162553.
[11]BIHARI P,VIPPOLA M,SCHULTES S,et al.Optimized dispersion of nanoparticles for biological in vitro and in vivo studies[J].Part Fibre Toxicol,2008,5(1):14-14.
[12]MOE B,GABOS S,LI X F.Real-time cell-microelectronic sensing of nanoparticle-induced cytotoxic effects[J].Anal Chim Acta,2013,789(14):83-90.
[13]张智勇.纳米毒理学研究方法与实验技术[M].北京:科学技术出版社,2015:157-177.
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