磁性三氧化二铁纳米粒子对小鼠腹腔巨噬细胞的氧化损伤作用(英文)

Oxidative injury of magnetic ferric oxide nanoparticles to peritoneal macrophage in mice

背景:已有报道表明纳米级的Fe2O3产生的细胞毒性与细胞的脂质过氧化存在一定的联系。氧化铁纳米粒子是否对巨噬细胞产生毒性,其毒性机制与氧化作用有何关联?目的:观察不同浓度Fe2O3纳米粒子对巨噬细胞的氧化损伤作用。设计:观察对比实验。单位:东南大学公共卫生学院。材料:RAW264.7细胞为小鼠腹腔巨噬细胞,购自中国科学院上海细胞所。Fe2O3纳米粒子(30nm)悬浮液由东南大学生物医学工程系制备提供。实验前先进行预处理:将Fe2O3纳米粒子悬浮液置60℃水浴中10h,然后37℃水浴过夜。如此反复3次,灭菌处理。小瓶分装,4℃保存。DMEM高糖培养液(美国Gibco公司);胰蛋白酶(美国Difco公司,进口分装);新生牛血清(杭州四季青公司);过氧化氢、羟自由基、超氧阴离子自由基、乳酸脱氢酶、超微量ATP酶和考马斯亮蓝蛋白含量测定试剂盒(南京建成生物技术公司)。方法:实验于2006-03/07在东南大学公共卫生学院劳动卫生与环境卫生学系实验室完成。RAW264.7细胞用DMEM培养基于37℃、体积分数为0.05的CO2培养箱中进行培养。每日用倒置显微镜观察细胞生长情况,取生长状态良好的对数生长期细胞进行试验。①细胞中氧自由基的检测:将密度为1.5×105L-1的巨噬细胞接种于24孔培养板,每孔1mL,37℃、体积分数为0.05的CO2培养箱中培养24h后,以质量浓度为1.0700、0.5350和0.2675g/L的Fe2O3纳米粒子(30nm)悬浮液染毒细胞为纳米粒子干预组,并设生理盐水为溶剂对照组,24h后终止培养,染毒结束后,低温条件下用玻璃匀浆器破碎细胞,按试剂盒说明,分别测定细胞中过氧化氢、羟自由基、超氧阴离子自由基。②培养液中乳酸脱氢酶活性及膜ATP酶活性的测定:纳米粒子干预组及对照组干预方法同上,染毒结束后,检测培养液中乳酸脱氢酶活性及膜ATP酶活性,乳酸脱氢酶活性的检测采用南京建成生物工程研究所提供的试剂盒,严格按照说明书进行操作。膜ATP酶活性的检测采用低温条件下用玻璃匀浆器破碎细胞,按试剂盒说明检测膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性。主要观察指标:①不同质量浓度Fe2O3纳米粒子对细胞过氧化氢、羟自由基和超氧阴离子的影响。②乳酸脱氢酶活性、膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性检测结果。结果:①Fe2O30.2675,0.5350,1.0700g/L纳米粒子干预组羟自由基水平高于溶剂对照组[(0.605±0.066),(0.410±0.080),(0.764±0.051),(0.285±0.057)mkat/g,P<0.05],超氧阴离子自由基高于溶剂对照组[(9.935±1.159),(8.912±0.131),(13.479±0.752),(5.635±0.475)μkat/g,P<0.05],Fe2O31.0700g/L过氧化氢水平高于溶剂对照组[(14.695±2.815),(2.397±0.399)mmol/L,P<0.05]②Fe2O3纳米粒子在实验浓度范围内,能够引起培养液中乳酸脱氢酶活性显著升高(P<0.05)。Fe2O3纳米粒子对细胞膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性均产生影响,且随着Fe2O3纳米粒子染毒剂量增加,Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性逐渐降低,与对照组比较,差异均具有显著性(P<0.05)。结论:Fe2O3纳米粒子剂量增加导致细胞内过氧化氢、羟自由基、超氧阴离子自由基增多,从而使细胞膜通透性增加,膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性受到抑制。

BACKGROUND ： Reports have demonstrated that cytotoxicity produced by ferric oxide （Fe2O3） nanoparticles is associated with cellular lipid peroxidation. Whether Fe2O3 nanoparticles have toxicity to macrophages, and what is the association of toxic mechanism and oxidization？ OB3ECTIVE： To observe the effects of different concentrations of Fe2O3 nanoparticles on the oxidative damage of macrophages. DESIGN ： A controlled observation experiment SEI-rING： School of Public Health, Southeast University MATERIALS： RAW264.7 cells were peritoneal macrophages of mouse and purchased from Shanghai Institute of calls, Chinese Academy of Sciences. Fe2O3 nanoparticles （30 nm） suspension was provided by Department of Biomedical Engineering, Southeast University）. Fe2O3 nanoparticle suspension was placed in 60 ℃ water for 10 hours, then in 37 ℃ water overnight. This procedure was repeated 3 times for germicidal treatment. Then, the suspension was packed into small bottles and stored at 4 ℃ for later use. DMEM high glucose culture fluid （Gibco Company, USA）; trypsinase （Difco Company, USA, imported）; new-born calf serum（Sijiqing Company, Hangzhou）; hydrogen dioxide （H2O2, Gibco Company）; Kits for measuring hydrogen dioxide（H2O2）, hydroxy radical （-OH）, superoxide anion radical （O2·^-）, lactic acid dehydrogenase, ultramicro ATP enzyme and Coomassie brilliant blue protein levels （Jiancheng Biotechnique Co., Ltd., Nanjing）, METHODS ： This experiment was carried out in the laboratory of Department of Labor and Environmental Health, School of Public Health, Dongnan University between March 2006 and July 2006. RAW264.7 cells （Abelson murine leukemia virus-induced tumor） were cultured in DMEM （Gibco Company） containing 100 g/L fetal bovine serum, 100 000 U/L penicillin and 100 mg/L streptomycin in the environment of 5% CO2. Cell growth was observed under an inverted microscope. Cells, which were at good exponential phase of growth, were chosen for test. ①Detection of oxygen free radical in the calls： 1,5x108 L-1 macrophages were inoculated to 24-well plate, 1 mL a wall. After the macrophages were cultured for 24 hours in incubation at 37 ℃ in a humidified atmosphere containing 5% CO2 1.070 0, 0.535 0 and 0.267 5 g/L Fe2O3 nanoparticles （30 nm） suspension-intervened macrophages were set as Fe2O3 nanoparticle group, and normal saline group was set as control group. Following the intervention of nanoparticles, macrophages ware disrupted With vitreous homogenizer.. The levels of hydrogen peroxide （H2O2）, hydroxyl radical （-OH） and superoxide anion （O2·^-） were determined in call lysates and supematants using the resgent kit （Nanjing Jiancheng Bioengineering Co., Ltd）. ② Determination of the activities of lactate dehydrogenase （LDH）, Na^＋ -K^＋ -ATPase and Ca^2＋ -Mg^2＋ -ATPase： Macrophages in the Fe2O3 nanoparticle group and control group were treated as above. The activities of LDH in culture medium ware determined according to the instruction of reagent kit （Nanjing Jiancheng Bioengineering Co,, Ltd）. And the activities of Na^＋ -K^＋ -ATPase and Ca^2＋ -Mg^2＋ -ATPase ware also determined according to the instruction of reagent kit （Nanjing Jiancheng Bioengineering Co., Ltd） at low temperature. MAIN OUTCOME MEASURES： ①Effects of different concentrations of Fe2O3 nanoparticles on the production of H2O2·OH and O2·^- in RAW264.7 cells. ②Effects of different concentrations of Fe2O3 nanoparticles on the activities of LDH , Na^＋ -K^＋ -ATPase and Ca^2＋ -Mg^2＋ -ATPase in RAW264.7 cell culture fluid. RESULTS; ① Level of -OH free radical in FeO3 nanoparticle 0.267 5, 0.535 0, 1.070 0 g/L groups was higher than that in control group, respectively [（0.605±0,066）, （0.410±0.080）, （0.764±0.051）, （0.285±0.057）mkat/g, P 〈 0,05]; Level of O2·^- free radical in Fe2O3 nanoparticle 0.267 5, 0.535 0, 1.070 0 g/L groups was higher than that in control group, respectively [（9,935±1.159）, （8.912＋0,131）, （13.479±0.752）, （5.635±0.475）μkat/g,P 〈 0.05]; Level of H2O2 in Fe2O3 nanoparticle 1.070 0 g/L group was higher than that in the control group [（14.695±2.815）, （2.397±0.399） mmol/L, P 〈 0.05].② Fe2O3 nanoparticles within the range of experimental concentration could cause LDH activity significantly increased （P 〈 0.05）. Fe2O3 nanoparticles had effects on the activities of Na^＋ ,K^＋ -ATPase and Ca^2＋,Mg^2＋ -ATPase. With the increase of dose of Fe2O3 nanoparticles, the activities of Na^＋ -K^＋ -ATPase and Ca^2＋ -Mg^2＋ -ATPase ware gradually decreased. There were significant differences as compared with control group （P 〈 0,05） CONCLUSION： Increasing dose of Fe2O3 nanoparticles would cause more H2O2,·OH and O2·^- free radicals in the calls, increase cell membrane permeability and inhibit the activities of LDH, Na^＋ K^＋ -ATPase and Ca^2＋ -Mg^2＋ -ATPase.