摘要
目的:研究比较不同长度和化学修饰的多壁碳纳米管(multi-walled carbon nanotubes,MWCNTs)对内皮细胞的活化作用,并探讨核苷酸结合寡聚结构域样受体家族含pyrin结构域蛋白3(nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3,NLRP3)炎性小体的相关机制。方法:采用动态光散射法对MWCNTs悬液进行表征,将不同长度[短MWCNT(short MWCNT,S-MWCNT)0.5~2.0μm或长MWCNT(long MWCNT,L-MWCNT)10~30μm]或相同长度(10~30μm)下不同化学修饰的MWCNTs[未修饰(L-MWCNT)、羧基修饰(L-MWCNT-COOH)、氨基修饰(L-MWCNT-NH2)和羟基修饰(L-MWCNT-OH)]作用于小鼠脑微血管内皮细胞系b.End3。分别采用细胞增殖检测(cell counting kit,CCK)-8试验和乳酸脱氢酶释放试验评价MWCNTs的细胞毒性,通过酶联免疫吸附试验测定胞外血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)含量并进一步使用人单核细胞系THP-1进行黏附力试验,评价不同种类MWCNTs的内皮细胞活化效应。进一步选择S-MWCNT、L-MWCNT和L-MWCNT-COOH探讨内皮细胞活化的炎症机制,采用免疫蛋白印迹法检测MWCNTs作用后细胞炎性小体蛋白NLRP3的表达水平。结果:在较高浓度(125μg/cm2)下作用24 h,不同种类的MWCNTs均可显著抑制b.End3的细胞活性并影响细胞膜完整性。在无明显细胞毒性的浓度下(6.25μg/cm2),不同种类的MWCNTs作用12 h均可显著诱导b.End3细胞内皮细胞活化,表现为VCAM-1释放水平增加,以及b.End3对THP-1的黏附水平增强。相同浓度下,L-MWCNT诱导细胞内皮细胞活化的效应显著强于S-MWCNT;相同长度下,L-MWCNT和L-MWCNT-COOH对细胞内皮细胞活化的效应差异无统计学意义。在6.25μg/cm2浓度下,S-MWCNT、L-MWCNT、L-MWCNT-COOH可时间依赖性地上调b.End3细胞的NLRP3蛋白表达水平。与相同浓度的S-MWCNT相比,L-MWCNT可显著上调细胞NLRP3的蛋白表达水平;在相同长度下,L-MWCNT和L-MWCNT-COOH作用后细胞的NLRP3蛋白
Objective:To investigate the effects of multi-walled carbon nanotubes(MWCNTs)with different length or chemical modification on endothelial cell activation and to explore the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome.Methods:MWCNTs were characterized by dynamic light scattering(DLS)after being suspended in culture medium.The immortalized mouse cerebral microvascular endothelial cell line b.End3 was treated with short MWCNTs(S-MWCNT,0.5 to 2μm),long MWCNTs(L-MWCNT,10 to 30μm)and the above long MWCNTs functionalized by carboxyl-(L-MWCNT-COOH),amino-(L-MWCNT-NH2)or hydroxyl-(L-MWCNT-OH)modification.Cytotoxicity of MWCNTs in b.End3 cells was determined by cell counting kit-8(CCK-8)assay and lactate dehydrogenase(LDH)release assay,and non-toxic low dose was selected for subsequent experiments.Effects of all types of MWCNTs on the endothelial activation of b.End3 were determined by the measurement of vascular cell adhesion molecule-1(VCAM-1)concentration in cell supernatant and adhesion assay of human monocytic cell line THP-1 to b.End3.To further elucidate the mechanism involved,the protein expressions of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)in cells treated with S-MWCNT,L-MWCNT and L-MWCNT-COOH were measured by Western blot.Results:At a higher concentration(125μg/cm2)and treated for 24 h,all types of MWCNTs significantly inhibited viability of b.End3 cells.At a sub-toxic concentration(6.25μg/cm2),all types of MWCNTs treated for 12 h significantly induced the activation of b.End3 cells,as evidenced by the elevated VCAM-1 release and THP-1 adhesion.Compared with S-MWCNT,L-MWCNT significantly promoted endothelial cell activation.L-MWCNT and L-MWCNT-COOH activated b.End3 cells to a similar extent.Furthermore,treatment with S-MWCNT,L-MWCNT and L-MWCNT-COOH increased NLRP3 expression in a time-dependent manner at 6.25μg/cm2.Compared with S-MWCNT,cells treated with L-MWCNT for 4 h and 12 h
作者
申杰
杨迪
陈梦圆
郭新彪
SHEN Jie;YANG Di;CHEN Meng-yuan;GUO Xin-biao(Department of Occupational and Environmental Health, Peking University School of Public Health, Beijing 100191, China)
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2021年第3期439-446,共8页
Journal of Peking University:Health Sciences
基金
国家自然科学基金(21477004)。
关键词
多壁碳纳米管
内皮细胞活化
长度
炎性小体
Multi-walled carbon nanotubes
Endothelial cell activation
Length
Inflammasome
