微摩尔浓度铜离子通过刺激活性氧产生促进人角质形成细胞增殖  

Micromolar concentration of copper( ii) ions enhances proliferation in human skin keratinocyte through appropriating generation of reactive oxygen species

作　　者：段小冬[1] 彭代智[1] 张宜澜[1] 陈志勇[1] 黄亚兰[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所创伤、烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2015年第11期1053-1058,共6页Journal of Third Military Medical University

基　　金：国家重点实验室自主研究课题Ⅰ类(SKLZZ201226)~~

摘　　要：目的观察不同浓度Cu2+对人角质形成细胞株(Ha Ca T)增殖活性及细胞内活性氧(reactive oxygen species,ROS)水平的影响,用活性氧清除剂N-乙酰半胱氨酸(NAC)验证两者之间的关系。方法不同浓度Cu2+(0、10-8、10-7、10-6、10-5、10-4、10-3mol/L)组或(和)5 mmol/L NAC处理Ha Ca T细胞后,采用CCK-8法和DCFH-DA分别检测细胞的增殖活性和细胞内ROS产生,共聚焦显微镜观察细胞内ROS产生及其与线粒体的分布情况。结果与阴性对照组(0 mol/L Cu2+)相比,10-6、10-5mol/L组Cu2+处理48 h后Ha Ca T细胞相对增殖率分别升高至116.19%(P=0.027)、113.37%(P=0.036),而其细胞内ROS产生在处理后30 min达到127.47%(P=0.001)、124.28%(P=0.031),60 min进一步上升至140.47%(P=0.002)、134.29%(P=0.007)。5 mmol/L NAC能拮抗微摩尔浓度Cu2+的上述作用。虽然10-8、10-7mol/L Cu2+在处理后60 min也刺激细胞内ROS产生明显升高至110.99%(P=0.016)及117.34%(P=0.016),但是处理48 h后细胞相对增殖率仅为101.09%和101.86%,并无促进细胞增殖的效应(P>0.05)。结论微摩尔浓度Cu2+能促进人角质形成细胞增殖,这种促增殖效应可能与刺激细胞内ROS水平的适度上调有关。Objective To determine the effects of micromolar copper （ii） ions （ Cu^2＋） on the proliferation and the generation of reactive oxygen species （ROS） in human skin keratinocyte line HaCaT, and verify their relationship through ROS scavenger, N-Acetyl-L-cysteine （NAC）. Methods HaCaT cells were treated with different concentrations of copper （ii） ions （0, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4 and 10^-3 tool/L） in presence or absence of 5 mmol/L NAC. Cell counting kit-8 （ CCK-8 ） assay and fluorescent probe DCFH-DA were used to measure the cell proliferation and ROS generation in the HaCaT cells, respectively. Laser confocal microscopy was employed to observe the intracellular ROS levels and mitochondria localization in the cells. Results After cultured with 10^-6and 10^-5mol/L Cu2＋ for 48 h, the cell relative proliferation rates reached 116.19% （P=0.027） and 113.37% （P =0. 036） respectively, and were significantly higher than that in negative control ceils （0 mol/L Cu^2＋ ）. Meanwhile, after exposure to 10^-6 and 10^-5 mol/L Cu^2＋ , the generation of intracellular ROS was remarkably increased to 127.47% （ P = 0. 001 ） and 124.27% （ P = 0.031） at 30 min, and 140.47% （P =0.002） and 134.29% （P =0.007） at 60 rain respectively. In addition, 5 mmol/L NAC significantly antagonized the cell proliferation and ROS generation induced by Cu2＋.Although intracellular ROS generation was also increased to 110.99% （P = 0. 016） and 117.34% （P = 0. 016） after 60 rain exposure under 10^-8and 10^-7mol/L Cu^2＋, the cell relative proliferation rates were only 101.09% and 101.86%, and no obvious change was observed in keratinocyte proliferation in these 2 groups and blank ceUs （ both P 〉 0.05 ）. Gonelusion Micromolar copper （ii） ions promote the proliferation of human skin keratinocyte through up-regulating ROS generation at moderate level.

关 键 词：铜离子 人角质形成细胞 细胞增殖 活性氧 N-乙酰半胱氨酸 

分 类 号：Q58[生物学—生物化学] R329.28[医药卫生—人体解剖和组织胚胎学]