不同修饰多壁碳纳米管诱导的细胞毒性及内质网相关基因表达

The cytotoxicity and expression changes of endoplasmic reticulum related genes induced by MWCNTs with different surface modifications

目的:比较表面不同修饰的两种多壁碳纳米管,即羧基化多壁碳纳米管(carboxylic multi-walled carbonnanotubes,c-MWCNTs)和牛磺酸修饰的多壁碳纳米管(taurine-modified multi-walled carbon nanotubes,tau-MWCNTs)对RAW264.7细胞的毒性,初步探究内质网在MWCNTs诱导细胞凋亡中的作用。方法:用尺寸和杂质含量一致的tau-MWCNTs和c-MWCNTs以0、1.56、3.12、6.25、12.50、25.00μg/cm2剂量染毒RAW264.7细胞3、6、12、24 h,通过WST-1法和AnnexinV-FITC/PI双染,检测细胞毒性和细胞凋亡。应用实时荧光定量PCR技术,检测与内质网钙离子调控、应激和凋亡相关的CRT、GRP78以及CHOP的mRNA表达水平。结果:tau-MWCNTs在所有的染毒剂量和染毒时间内,细胞毒性均比c-MWCNTs低。在≥12.50μg/cm2剂量下染毒3 h以上,或在≥3.12μg/cm2剂量下染毒12 h以上,两种MWCNTs均能产生明显的细胞毒性且差异有统计学意义(P<0.05)。凋亡检测发现,两种MWCNTs染毒细胞24 h的细胞凋亡率显著高于12 h,且c-MWCNTs组的凋亡率普遍高于tau-MWCNTs组。实时荧光定量PCR结果显示,在所研究的染毒剂量及时间内,CRT、GRP78和CHOP mRNA表达与对照相比差异很小,无统计学意义。结论:tau-MWCNTs对RAW264.7细胞的毒性较c-MWCNTs明显降低,未观察到MWCNTs对RAW264.7细胞内质网造成损伤,提示内质网通路可能不是MWCNTs导致细胞凋亡的主要机制。

Objective：To compare the cytotoxicity of RAW264.7 cells induced by two types of multi-walled carbon nanotubes（MWCNTs） with different surface modifications（MWCNTs modified by taurine and MWCNTs treated by acid）,and explore the role of endoplasmic reticulum（ER） in MWCNTs-induced apoptosis.Methods：RAW264.7 cells were exposed to tau-MWCNTs or c-MWCNTs,of which the diameters and impurity contents were the same,at the dose of 0,1.56,3.12,6.25,12.50,25.00 μg/cm2 for 3,6,12 and 24 h,respectively.Then the cytotoxicity of MWCNTs was determined by WST-1 assay,and the percentages of apoptosis were analyzed via flow cytometry with AnnexinV-FITC/PI label.Real-time PCR was used to detect mRNA expression levels of CRT,GRP78 and CHOP genes which were related to ER calcium regulation,stress and apoptosis.Results：Compared with the c-MWCNTs groups,the cytotoxicity of tau-MWCNTs was lower at the same dose and treatment time.Both the two types of MWCNTs could induce cytotoxicity apparently and statistically,when the cells were treated for only 3 h at the doses of more than 12.50 μg/cm2 or treated for more than 12 h at the lower dose of 3.12 μg/cm2（P〈0.05）.The results of flow cytometry showed that the two MWCNTs could induce apoptosis of RAW264.7 cells in the dose-and time-dependently manner.The apoptosis rate of the cells treated for 24 h was higher than that for 12 h.And at each dose,the apoptosis rate induced by c-MWCNTs was also higher than that of the water soluble tau-MWCNTs within our study design.However,in this study,the mRNA expression levels of CRT,GRP78 and CHOP mRNA treated with both types of MWCNTs did not show any significant differences compared with the control groups.Conclusion：The cytotoxicity of tau-MWCNTs was much lower than that of c-MWCNTs.Unfortunately,we found the expression of genes related to ER had little effects on the apoptosis of RAW264.7 treated with MWCNTs,indicating that the ER pathway might not be the mechanism of MWCNTs-induced apoptosis.