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(P<0.01).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(P<0.01).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(P<0.01),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(P<0.01),但溶酶体亮度增加,且HLF细胞LC3B的含量显著升高(P<0.01);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.%OBJECTIVE To study the cytotoxic characteristics of nitrogen mustard HN-3 in different cell. METHODS Human epidermal keratinocytes-fetal (HEKf), human dermal fibroblasts-adult (HDFa) and human lung fibroblasts (HLF) cell lines were treated with HN-3100, 300 and 450μmol·L-1 for 0.5, 2, 4, 6, 12, 24 and 48 h, respectively. Multi-parameter analysis technology based on cell imaging was used to examine the effects of HN-3 on cell survival, cell cycle arrest, apoptosis, autophagy and oxidative stress, along with parameters concerning nucleus, cytoskeleton (actin and tubulin), lysosome, nuclear membrane permeability (NMP), mitochondrial membrane potential (MMP) and phosphohistone H 2AX (pH2AX). RESULTS HN-3 caused irreversible cellular damage by significantly decreasing the number of HEKf, HDFa and HLF cells in a time-dependent manner (P<0.01). Before the cell number was reduced robustly, the content of reactive oxygen species and pH2AX significantly increased, but the glutathione content decreased after cells were exposed to HN-3 for 0.5 h (P<0.01). In addition, the content of lyso-some was reduced in HEKf cells at 0.5 h, but increased in HDFa and HLF cells at 0.5 and 2 h respec-tively, accompanied by the increase in microtubule-associated protein 1 light chain 3B (LC3B) puncta.With the significant reduction of the cell number in HEKf cell line, the nuclear intensity increased, nuclear area decreased, the intensity and area of F-actin and α-tubulin decreased, MMP decreased (P<0.01) and lysosomal intensity increased. But the effects of HN-3 on HDFa and HLF cell lines were quite different. The nuclear area increased, the intensity and area of F-actin and a-tubulin increased, MMP increased (P<0.01) and the intensity of lysosome increased. In HLF cells, there was an increase in LC3B puncta (P<0.01). In all the three cell lines, NMP and manganese superoxide dismutase content were increased, and cell cycle arrested at G2 phase. HN-3 Induced early apoptosis in HDFa cells but late apoptosis in HEKf cells. CONCLUSION HN-3 causes DNA damage, oxidative stress and lysosome damage at an early stage, whereas at the late stage, the imbalance of MMP, increase in NMP, and G2 phage arrest are the major cytotoxic effects. Moreover, HN-3 specifically induces nuclear condensation, cytoskeleton protein aggregation and apoptosis in HEKf cell. HN-3 Induces nuclear swelling, and loose cytoskeleton in HDFa cells and HLF cells, eventually inducing early apoptosis in HDFa cells and autophagic death in HLF cells.
