不同粒径SiO_2颗粒诱导BEAS-2B细胞凋亡的研究
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摘要
目的探讨不同粒径二氧化硅(silica particles,SiO_2)颗粒对支气管上皮细胞凋亡的诱导作用,进一步阐明SiO_2颗粒粒径相关的毒作用机制。方法以体外培养的支气管上皮细胞(Bronchial epithelial cells,BEAS-2B)为模型,随机分为对照组和SiO_2(Nano-Si40、Nano-Si60和Si200)颗粒暴露组,暴露浓度为6.25、12.5和25μg/ml。采用透射电子显微镜(TEM)观察3种SiO_2颗粒粒径、形貌特征及分散稳定性。细胞处理24 h后,采用MTT比色法测定细胞存活率;采用乳酸脱氢酶(lactate dehydrogenase,LDH)法测定细胞膜的完整性;TEM观察SiO_2颗粒摄取;采用倒置荧光显微镜观察4',6-二脒基-2-苯基吲哚(DAPI)染色后细胞核的形态;Annexin V/PI双染标记后采用流式细胞术(flow cytometry,FCM)检测细胞凋亡的情况。结果电子显微镜结果显示3种SiO_2颗粒分布均匀,大小均一,颗粒呈圆形或椭球形,分散性良好,未发生明显聚集。Image J软件计算颗粒平均粒径分别为41.26±3.78、61.51±7.93和206.31±18.70 nm。与对照组相比,SiO_2颗粒作用BEAS-2B细胞24 h后,细胞存活率下降(P<0.05);细胞培养液中LDH活力明显升高(P<0.05);3种SiO_2颗粒均能够被BEAS-2B细胞摄取,大量的粒子以单个粒子或以膜包裹的聚集状态分布在胞质中,呈核周分布的现象;DAPI染色细胞核出现核固缩、核碎裂和染色质凝聚边缘化等典型的凋亡细胞核形态;3种SiO_2颗粒均能诱导细胞凋亡发生,且随着颗粒浓度的增加和粒径的减小,凋亡率逐渐升高。结论 SiO_2颗粒可被BEAS-2B细胞摄取,降低细胞存活率,进而破坏细胞膜完整性,最终诱导细胞凋亡发生,且具有明显的剂量和粒径依赖趋势。
Objective To investigate the apoptosis induced by different sizes of silica particles( SiO_2: Nano-Si40,Nano-Si60 and Si200) on Bronchial epithelial cells,and to further clarify the size related toxicity mechanisms induced by SiO_2. Methods The cultured Bronchial epithelial cells( BEAS-2 B) were utilized as cell model in vitro,and divided into control and SiO_2 exposure groups( Nano-Si40,Nano-Si60 and Si200) randomly,with the concentrations of 6. 25,12. 5 and 25 μg/ml respectively. Employing the transmission electron microscope( TEM) to determine particle size,morphology and dispersion of SiO_2. After 24 h exposure,utilizing MTT assay to detect the cell viability. Membrane integrity were detected by lactate dehydrogenase( LDH) release assay. Employing the TEM to detect whether cellular uptake was induced by SiO_2. Using the fluorescence microscope to observe the nuclear morphology after DAPI staining. Employing the flow cytometry( FCM) to measure apoptosis by using Annexin V-FITC/PI fluorescent double labeling. Result The TEM images illustrated that SiO_2 was nearly spherical with uniform size and dispersed well. The average particle sizes were 41. 26 ± 3. 78 nm,61. 51 ± 7. 93 nm and206. 31 ± 18. 70 nm calculated by using image J software,respectively. Compared with the control group,cell viability in SiO_2 exposure groups was inhibited after 24 h( P < 0. 05). LDH release in medium were increased( P < 0. 05). TEM observation showed that three sizes of SiO_2 can enter BEAS-2 B cells and mainly appeared in dispersed or aggregate form in the cytoplasm,with distribution around nuclear peripheny distributed phenomenon. DAPI staining of nuclei showed that typical apoptotic nuclear morphology. Flow cytometry detection showed that three sizes of SiO_2 induced apoptosis in BEAS-2 B cells( P < 0. 05). Conclusion The result indicated that SiO_2 could be ingested BEAS-2 B cells,induce loss of cell viability,damage membrane integrity,and apoptosis ultimately,with a dose-and size-dependent manner.
Objective To investigate the apoptosis induced by different sizes of silica particles( SiO_2: Nano-Si40,Nano-Si60 and Si200) on Bronchial epithelial cells,and to further clarify the size related toxicity mechanisms induced by SiO_2. Methods The cultured Bronchial epithelial cells( BEAS-2 B) were utilized as cell model in vitro,and divided into control and SiO_2 exposure groups( Nano-Si40,Nano-Si60 and Si200) randomly,with the concentrations of 6. 25,12. 5 and 25 μg/ml respectively. Employing the transmission electron microscope( TEM) to determine particle size,morphology and dispersion of SiO_2. After 24 h exposure,utilizing MTT assay to detect the cell viability. Membrane integrity were detected by lactate dehydrogenase( LDH) release assay. Employing the TEM to detect whether cellular uptake was induced by SiO_2. Using the fluorescence microscope to observe the nuclear morphology after DAPI staining. Employing the flow cytometry( FCM) to measure apoptosis by using Annexin V-FITC/PI fluorescent double labeling. Result The TEM images illustrated that SiO_2 was nearly spherical with uniform size and dispersed well. The average particle sizes were 41. 26 ± 3. 78 nm,61. 51 ± 7. 93 nm and206. 31 ± 18. 70 nm calculated by using image J software,respectively. Compared with the control group,cell viability in SiO_2 exposure groups was inhibited after 24 h( P < 0. 05). LDH release in medium were increased( P < 0. 05). TEM observation showed that three sizes of SiO_2 can enter BEAS-2 B cells and mainly appeared in dispersed or aggregate form in the cytoplasm,with distribution around nuclear peripheny distributed phenomenon. DAPI staining of nuclei showed that typical apoptotic nuclear morphology. Flow cytometry detection showed that three sizes of SiO_2 induced apoptosis in BEAS-2 B cells( P < 0. 05). Conclusion The result indicated that SiO_2 could be ingested BEAS-2 B cells,induce loss of cell viability,damage membrane integrity,and apoptosis ultimately,with a dose-and size-dependent manner.
引文
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[2]Eastlake AC,Beaucham C,Martinez KF,et al.Refinement of the nanoparticle emission assessment technique into the nanomaterial exposure assessment technique(NEAT 2.0)[J].J Occup Environ Hyg,2016,13(9):708-717.
[3]Lin C,Zhao X,Sun D,et al.Transcriptional activation of follistatin by Nrf2 protects pulmonary epithelial cells against silica nanoparticle-induced oxidative stress[J].Sci RepUk,2016,6(5):21133.
[4]Bakand S,Hayes A,Dechsakulthorn F.Nanoparticles:a review of particle toxicology following inhalation exposure[J].Inhal Toxicol,2012,24(2):125-135.
[5]Ma J,Mercer RR,Barger M,et al.Effects of amorphous silica coating on cerium oxide nanoparticles induced pulmonary responses[J].Toxicol Appl Pharm,2015,288(1):63-73.
[6]Duan J,Yu Y,Li Y,et al.Cardiovascular toxicity evaluation of silica nanoparticles in endothelial cells and zebrafish model[J].Biomaterials,2013,34(23):5853-5862.
[7]Maser E,Schulz M,Sauer UG,et al.Invitro and in vivo genotoxicity investigations of differently sized amorphous Si O2nanomaterials[J].Mutat Res-Gen Tox En,2015,9(1):177-203.
[8]Yu Y,Li Y,Wang W,et al.Acute toxicity of amorphous silica nanoparticles in intravenously exposed ICR mice[J].Plo S One,2013,8(4):e61346.
[9]Nabeshi H,Yoshikawa T,Matsuyama K,et al.Sizedependent cytotoxic effects of amorphous silica nanoparticles on Langerhans cells[J].Pharmazie,2010,65(3):199-201.
[10]Du Z,Zhao D,Jing L,et al.Cardiovascular toxicity of different sizes amorphous silica nanoparticles in rats after intratracheal instillation[J].Cardiovasc Toxicol,2013,13(3):194-207.
[11]Gong C,Tao G,Yang L,et al.The role of reactive oxygen species in silicon dioxide nanoparticle-induced cytotoxicity and DNA damage in Ha Ca T cells[J].Mol Biol Rep,2012,39(4):4915-4925.
[12]夏银叶,李艳博,牛丕业,等.纳米二氧化硅颗粒对血管内皮细胞的毒性及其凋亡诱导作用[J].吉林大学学报(医学版),2015,41(3):454-459.
[13]Li Y,Sun L,Jin M,et al.Size-dependent cytotoxicity of amorphous silica nanoparticles in human hepatoma Hep G2cells[J].Toxicol In Vitro,2011,25(7):1343-1352.
[14]Kasper J,Hermanns MI,Bantz C,et al.Interactions of silica nanoparticles with lung epithelial cells and the association to flotillins[J].Arch Toxicol,2013,87(6):1053-1065.
[15]Duan J,Yu Y,Li Y,et al.Toxic effect of silica nanoparticles on endothelial cells through DNA damage response via Chk1-dependent G2/M checkpoint[J].Plo S One,2013,8(4):e62087.