活性氧、丙二醛及超氧化物歧化酶在NaAsO_2致人角质形成细胞恶性转化过程中的动态变化  

Dynamic changes of ROS,MDA and SOD during arsenic-induced neoplastic transformation in human keratinocytes

作　　者：马园[1] 王大朋[1] 许熙国[1] 杨旭[1] 王小娟[1] 朱震[1] 赵莹莹[1] 陈敏[1] 许菲菲[1] 付乐[1] 朱继刚[1] 高芬芳[1] 安艳[1] 

机构地区：[1]苏州大学医学部公共卫生学院卫生毒理教研室,苏州215123

出　　处：《卫生研究》2015年第3期456-461,共6页Journal of Hygiene Research

基　　金：高等学校博士学科点专项科研基金-博导类(No.20123201110012);江苏省普通高校研究生科研创新计划(No.ZY32007613);苏州大学国家自然科学基金预研项目(No.Q3126982)

摘　　要：目的探讨氧化应激相关指标活性氧(ROS)、丙二醛(MDA)和超氧化物歧化酶(SOD)在砷致人角质形成细胞(human keratinocytes,Ha Ca T)恶性转化不同阶段的动态变化。方法以含1.0μmol/L Na As O2的DMEM高糖完全培养基连续培养Ha Ca T细胞35代后,用基质金属蛋白酶-9(MMP-9)表达水平变化、细胞生长动力学改变和软琼脂集落形成实验鉴定其是否发生恶性转化。分别收集0、1、7、14、21、28和35代细胞,采用流式细胞仪检测不同代数细胞内ROS活性,生化法检测细胞内MDA含量及SOD活性变化。结果 1.0μmol/L Na As O2处理Ha Ca T细胞28代开始MMP-9相对蛋白水平明显上升,35代后细胞生长速度明显增快,倍增时间明显缩短;软琼脂集落形成数为(107±11)个,较对照组(24±7)明显增多(P<0.01)。ROS水平在染毒0到14代间呈先上升后下降随后再上升趋势,14代以后ROS水平逐渐降低。MDA含量在染毒1代时达到峰值,随后呈缓慢下降趋势。SOD活性在染毒0到21代呈先上升后缓慢下降趋势,21代以后其活性再次升高,呈持续上升趋势,且较0代明显升高(P<0.05)。结论低剂量亚砷酸钠长期诱导Ha Ca T细胞发生恶性转化过程中伴随细胞内氧化-抗氧化平衡失调,染毒后期SOD活性持续增加而ROS水平持续降低。Objective To investigate tile level of ROS, MDA and SOD in different stages of arsenic-induced neoplastic transformation in human keratinocytes. Methods HaCaT human immortalized keratinocytes were continuously exposed to 1.0 μmol/L arsenite for 35 passages. The secretion of active MMP-9, the proliferation rate and doubling time nd colony formation assay in soft agar colony were used to identify the malignant phenotype of the arsenite-exposed HaCaT cells. Then flow cytometry was used to detect the levels of ROS, and the level of MDA and SOD were tested by biochemical method at different passages of arsenite exposure in HaCaT cells. Results A marked increase in the secretion of active MMP-9 in the arsenic-treated （ 1.0 μmol/L NaAsO2） cells was observed in comparison to the passage-matched untreated control （0.0 μmol/L NaAsO2） cells at 28 and 35 passages. And compared with 0. 0 μmol/L NaAsO2group, the proliferation rate and doubling time in 1.0 μmol/L NaAsO2 group was much faster at 21 （ （64. 37 ± 15.92）h） and 28（ （64.04 ± 12.84） h） passages with a significant statistical difference at passage 35 （ （54.00 ± 2.35 ） h） （ P 〈 0.05 ）. Furthermore, the long-term arsenite-treated cells formed significantly higher colonies（ 107 ± 11 in passage 35 ） in soft agar than control cells （ P 〈 0.05 ）. No obvious regularity changes of ROS and MDA levels were found before 14 passages of arsenite exposure, except for passage 1. Surprisingly, after 14 passages, with the increased passages of exposure to arsenite, both the ROS and MDA levels decreased gradually, the ROS level at passage 55 was significant lower compared to passage 0 （P 〈 0.05 ）. Conversely, after passage 21, the activity of SOD was obviously enhanced and reached the highest level at passage 35. Conclusion Long-term exposure to low concentrations of inorganic arsenic-induced malignant transformation of HaCaT cells is accompanied by intracellular imbalance between oxidative-antioxidant, which increased expres

关 键 词：亚砷酸盐 HACAT细胞 恶性转化 氧化应激 

分 类 号：R994.6[医药卫生—毒理学] R730.261[医药卫生—药学]