基于细胞高内涵分析表征氮芥HN-3的细胞毒性

发布时间：2018-06-26 19:53

本文选题：氮芥 + 细胞毒表型　；参考：《中国药理学与毒理学杂志》2017年07期

【摘要】：目的研究氮芥HN-3对不同来源细胞的毒性表型特征。方法 HN-3100,300和450μmol·L~(-1)分别作用于原代人胚表皮角质细胞(HEKf),原代成人皮肤成纤维细胞(HDFa)和人肺成纤维细胞(HLF)0.5,2,4,6,8,24和48 h后,采用基于细胞成像的多参数分析技术,检测HN-3对细胞存活、细胞核、细胞骨架(微丝和微管)、线粒体膜电位(MMP)、氧化应激、核膜通透性(NMP)、磷酸化组蛋白、溶酶体、自噬、细胞周期和凋亡等参数的影响。结果 HN-3引起不可逆性的细胞损伤,表现为存活细胞数量显著减少(P0.01)。在存活细胞数量显著减少前,从0.5 h开始,细胞内活性氧和磷酸化组蛋白水平即显著升高,谷胱甘肽含量显著降低(P0.01)。HEKf细胞内溶酶体在0.5 h时减少,而HDFa和HLF细胞内溶酶体则分别在0.5和2 h升高,并伴有微管相关蛋白1轻链3B(LC3B)蛋白表达升高伴随存活细胞数量减少,HEKf细胞核亮度升高,核面积减少,微丝、微管亮度和面积减少,MMP显著降低(P0.01),溶酶体亮度有增加趋势;与此相反,HDFa和HLF细胞表现为核面积增加,微丝、微管亮度和面积增加,MMP显著升高(P0.01),但溶酶体亮度增加,且HLF细胞LC3B的含量显著升高(P0.01);同时,3种细胞均有NMP增高和锰超氧化物歧化酶含量增加,G2期阻滞。此外,HEKf细胞出现早期和晚期凋亡,HDFa细胞出现早期凋亡。结论 HN-3诱发早期的细胞损伤效应包括DNA损伤、氧化胁迫和溶酶体损伤,晚期导致MMP失衡、NMP增加、G2期细胞周期阻滞;此外,HN-3特异性地诱导HEKf细胞核固缩、细胞骨架蛋白核聚集和细胞凋亡;诱导HDFa和HLF细胞核肿胀和细胞骨架松散,并最终导致HDFa细胞早凋和HLF细胞自噬性死亡。
[Abstract]:Objective to study the toxic phenotypic characteristics of nitrogen mustard HN-3 on different origin cells. Methods HN-3100300 and 450 渭 mol L ~ (-1) were applied to primary human embryonic keratinocytes (HEKf), adult skin fibroblasts (HDFa) and human lung fibroblasts (HLF) for 24 and 48 hours, respectively. Effects of nuclear, cytoskeleton (microfilaments and microtubules), mitochondrial membrane potential (MMP), oxidative stress, nuclear membrane permeability (NMP), phosphorylated histone, lysosome, autophagy, cell cycle and apoptosis. Results HN-3 induced irreversible cell damage, showing a significant decrease in the number of viable cells (P0.01). Before the number of surviving cells decreased significantly, the levels of reactive oxygen species and phosphorylated histone increased significantly at 0.5 h, and the content of glutathione decreased significantly (P0.01). The lysosomes in HEKf cells decreased at 0.5 h. However, the lysosomes in HDFa and HLF cells increased at 0.5 h and 2 h, respectively, and accompanied by the increase of microtubule-associated protein 1 light chain 3B (LC3B) protein expression. The brightness and area of microtubules decreased significantly (P0.01), and the brightness of lysosome increased. On the contrary, HDFa and HLF cells showed increased nuclear area, microfilament, microtubule brightness and area increased significantly (P0.01), but lysosomal brightness increased. The content of LC3B in HLF cells was significantly increased (P0.01), and the increase of NMP and the increase of manganese superoxide dismutase (MSOD) in HLF cells were blocked in G2 phase. In addition, early and late apoptosis occurred in HEKf cells and early apoptosis in HDFa cells. Conclusion the early cell damage effects induced by HN-3 include DNA damage, oxidative stress, lysosome damage, late stage MMP imbalance and NMP increasing cell cycle arrest in G 2 phase, in addition, HN-3 specifically induces nuclear pyknosis in HEKf. Nuclear aggregation and apoptosis of cytoskeleton protein induced swelling of HDFa and HLF nuclei and loosening of cytoskeleton, which resulted in early apoptosis of HDFa cells and autophagic death of HLF cells.
【作者单位】：抗毒药物与毒理学国家重点实验室;军事医学科学院毒物药物研究所;
【基金】：国家自然科学基金(81430090) 综合性新药研究开发关键技术(2012ZX09301003-001);综合性新药研究开发关键技术(2012ZX09301003-003)~~
【分类号】：R99

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