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(30 nm)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10 h,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1.5×105 L-1的巨噬细胞接种于24孔培养板,每孔1 mL,37 ℃、体积分数为0.05的CO2培养箱中培养24 h后,以质量浓度为1.0 700、0.5 350和0.2 675 g/L的Fe2O3纳米粒子(30 nm)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,24 h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:①Fe2O3 0.267 5,0.535 0,1.070 0 g/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],Fe2O3 1.070 0 g/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.
揹景:已有報道錶明納米級的Fe2O3產生的細胞毒性與細胞的脂質過氧化存在一定的聯繫.氧化鐵納米粒子是否對巨噬細胞產生毒性,其毒性機製與氧化作用有何關聯?目的:觀察不同濃度Fe2O3納米粒子對巨噬細胞的氧化損傷作用.設計:觀察對比實驗.單位:東南大學公共衛生學院.材料:RAW264.7細胞為小鼠腹腔巨噬細胞,購自中國科學院上海細胞所.Fe2O3納米粒子(30 nm)懸浮液由東南大學生物醫學工程繫製備提供.實驗前先進行預處理:將Fe2O3納米粒子懸浮液置60 ℃水浴中10 h,然後37 ℃水浴過夜.如此反複3次,滅菌處理.小瓶分裝,4 ℃保存.DMEM高糖培養液(美國Gibco公司);胰蛋白酶(美國Difco公司,進口分裝);新生牛血清(杭州四季青公司);過氧化氫、羥自由基、超氧陰離子自由基、乳痠脫氫酶、超微量ATP酶和攷馬斯亮藍蛋白含量測定試劑盒(南京建成生物技術公司).方法:實驗于2006-03/07在東南大學公共衛生學院勞動衛生與環境衛生學繫實驗室完成.RAW264.7細胞用DMEM培養基于37 ℃、體積分數為0.05的CO2培養箱中進行培養.每日用倒置顯微鏡觀察細胞生長情況,取生長狀態良好的對數生長期細胞進行試驗.①細胞中氧自由基的檢測:將密度為1.5×105 L-1的巨噬細胞接種于24孔培養闆,每孔1 mL,37 ℃、體積分數為0.05的CO2培養箱中培養24 h後,以質量濃度為1.0 700、0.5 350和0.2 675 g/L的Fe2O3納米粒子(30 nm)懸浮液染毒細胞為納米粒子榦預組,併設生理鹽水為溶劑對照組,24 h後終止培養,染毒結束後,低溫條件下用玻璃勻漿器破碎細胞,按試劑盒說明,分彆測定細胞中過氧化氫、羥自由基、超氧陰離子自由基.②培養液中乳痠脫氫酶活性及膜ATP酶活性的測定:納米粒子榦預組及對照組榦預方法同上,染毒結束後,檢測培養液中乳痠脫氫酶活性及膜ATP酶活性,乳痠脫氫酶活性的檢測採用南京建成生物工程研究所提供的試劑盒,嚴格按照說明書進行操作.膜ATP酶活性的檢測採用低溫條件下用玻璃勻漿器破碎細胞,按試劑盒說明檢測膜Na+-K+-ATP酶和Ca2+Mg2+-ATP酶活性.主要觀察指標:①不同質量濃度Fe2O3納米粒子對細胞過氧化氫、羥自由基和超氧陰離子的影響.②乳痠脫氫酶活性、膜Na+-K+-ATP酶和Ca2+Mg2*-ATP酶活性檢測結果.結果:①Fe2O3 0.267 5,0.535 0,1.070 0 g/L納米粒子榦預組羥自由基水平高于溶劑對照組[(0.605±0.066),(0.410±0.080),(0.764±0.051),(0.285±0.057)mkat/g,P＜0.05],超氧陰離子自由基高于溶劑對照組[(9.935±1.159),(8.912±0.131),(13.479±0.752),(5.635±0.475)μkat/g,P＜0.05],Fe2O3 1.070 0 g/L過氧化氫水平高于溶劑對照組[(14.695±2.815),(2.397±0.399)mmol/L,P＜0.05]②Fe2O3納米粒子在實驗濃度範圍內,能夠引起培養液中乳痠脫氫酶活性顯著升高(P＜0.05).Fe2O3納米粒子對細胞膜Na*-K*-ATP酶和Ca2+-Mg2+-ATP酶活性均產生影響,且隨著Fe2O3納米粒子染毒劑量增加,Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性逐漸降低,與對照組比較,差異均具有顯著性(P＜0.05).結論:Fe2O3納米粒子劑量增加導緻細胞內過氧化氫、羥自由基、超氧陰離子自由基增多,從而使細胞膜通透性增加,膜Na+-K*-ATP酶和Ca2+-Mg2+-ATP酶活性受到抑製.
배경:이유보도표명납미급적Fe2O3산생적세포독성여세포적지질과양화존재일정적련계.양화철납미입자시부대거서세포산생독성,기독성궤제여양화작용유하관련?목적:관찰불동농도Fe2O3납미입자대거서세포적양화손상작용.설계:관찰대비실험.단위:동남대학공공위생학원.재료:RAW264.7세포위소서복강거서세포,구자중국과학원상해세포소.Fe2O3납미입자(30 nm)현부액유동남대학생물의학공정계제비제공.실험전선진행예처리:장Fe2O3납미입자현부액치60 ℃수욕중10 h,연후37 ℃수욕과야.여차반복3차,멸균처리.소병분장,4 ℃보존.DMEM고당배양액(미국Gibco공사);이단백매(미국Difco공사,진구분장);신생우혈청(항주사계청공사);과양화경、간자유기、초양음리자자유기、유산탈경매、초미량ATP매화고마사량람단백함량측정시제합(남경건성생물기술공사).방법:실험우2006-03/07재동남대학공공위생학원노동위생여배경위생학계실험실완성.RAW264.7세포용DMEM배양기우37 ℃、체적분수위0.05적CO2배양상중진행배양.매일용도치현미경관찰세포생장정황,취생장상태량호적대수생장기세포진행시험.①세포중양자유기적검측:장밀도위1.5×105 L-1적거서세포접충우24공배양판,매공1 mL,37 ℃、체적분수위0.05적CO2배양상중배양24 h후,이질량농도위1.0 700、0.5 350화0.2 675 g/L적Fe2O3납미입자(30 nm)현부액염독세포위납미입자간예조,병설생리염수위용제대조조,24 h후종지배양,염독결속후,저온조건하용파리균장기파쇄세포,안시제합설명,분별측정세포중과양화경、간자유기、초양음리자자유기.②배양액중유산탈경매활성급막ATP매활성적측정:납미입자간예조급대조조간예방법동상,염독결속후,검측배양액중유산탈경매활성급막ATP매활성,유산탈경매활성적검측채용남경건성생물공정연구소제공적시제합,엄격안조설명서진행조작.막ATP매활성적검측채용저온조건하용파리균장기파쇄세포,안시제합설명검측막Na+-K+-ATP매화Ca2+Mg2+-ATP매활성.주요관찰지표:①불동질량농도Fe2O3납미입자대세포과양화경、간자유기화초양음리자적영향.②유산탈경매활성、막Na+-K+-ATP매화Ca2+Mg2*-ATP매활성검측결과.결과:①Fe2O3 0.267 5,0.535 0,1.070 0 g/L납미입자간예조간자유기수평고우용제대조조[(0.605±0.066),(0.410±0.080),(0.764±0.051),(0.285±0.057)mkat/g,P＜0.05],초양음리자자유기고우용제대조조[(9.935±1.159),(8.912±0.131),(13.479±0.752),(5.635±0.475)μkat/g,P＜0.05],Fe2O3 1.070 0 g/L과양화경수평고우용제대조조[(14.695±2.815),(2.397±0.399)mmol/L,P＜0.05]②Fe2O3납미입자재실험농도범위내,능구인기배양액중유산탈경매활성현저승고(P＜0.05).Fe2O3납미입자대세포막Na*-K*-ATP매화Ca2+-Mg2+-ATP매활성균산생영향,차수착Fe2O3납미입자염독제량증가,Na+-K+-ATP매화Ca2+-Mg2+-ATP매활성축점강저,여대조조비교,차이균구유현저성(P＜0.05).결론:Fe2O3납미입자제량증가도치세포내과양화경、간자유기、초양음리자자유기증다,종이사세포막통투성증가,막Na+-K*-ATP매화Ca2+-Mg2+-ATP매활성수도억제.
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?OBJECTIVE: To observe the effects of different concentrations of Fe2O3 nanoparticles on the oxidative damage of macrophages.DESIGN: A controlled observation experiment.SETTING: School of Public Health, Southeast University.MATERIALS: RAW264.7 cells were peritoneal macrophages of mouse and purchased from Shanghai Institute of cells, 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-bom 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 bovineserum, 100 000 U/L penicillin and 100 mg/L streptomycin in the environment of 5% CO2. Cell growth was observed under an inverted radical in the cells: 1.5×108 L-1 macrophages were inoculated to 24-well plate, 1 mLa well. After the macrophages were cultured for 24 hours in incubation at 37 ℃ in a humidified atmosphere containing 5% CO2. 1.070 0, 0.5350 and 0.2675 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 were disrupted with Determination of the activities of lactate dehydrogenase (LDH), Na+-K+-ATPase and Ca2+-Mg2+-ATPase: Macrophages in the Fe2O3 nanoparticle group and control group were treated as above. The activities of LDH in culture medium were determined according to the instruction of reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd). And the activities of Na+-K+-ATPase and Ca+-Mg2+-ATPase were 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 Ca2+-Mg2+-ATPase in RAW264.7 cell culture fluid.RESULTS: ① Level of ·OH 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 [(0.605±0.066), (0.410±0.080), (0.764±0.051), (0.285±0.057)mkat/g, P ＜ 0.05]; Level of 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 ＜increased (P ＜ 0.05). Fe2O3 nanoparticles had effects on the activities of Na+,K+-ATPase and Ca2+,Mg2+-ATPase. With the increase of dose of Fe2O3 nanoparticles, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were gradually decreased. There were significant differences as compared with control group (P ＜ 0.05)CONCLUSION: Increasing dose of Fe2O3 nanoparticles wouldcause more H2O2, ·OH and O2·- free radicals in the cells, increase cell membrane permeability and inhibit the activities of LDH, Na+-K+-ATPase and Ca2+-Mg2+-ATPase.
