钽颗粒影响成骨细胞增殖的机制研究
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摘要
目的研究钽颗粒对成骨细胞增殖的影响,并初步探索其机制。方法将不同浓度的微粒钽和纳米钽颗粒分别与成骨细胞共培养,CCK8法检测6、12、24、48 h时细胞的OD值。依据CCK8结果,筛选促增殖作用最明显的组别,用Western blot、激光共聚焦显微镜、透射电镜检测细胞自噬水平。最后,结合自噬诱导剂和自噬抑制剂进一步验证自噬在纳米钽促增殖反应中的作用。结果 100ng/mL微米钽处理组促增殖作用明显,但未检测到细胞自噬;20μg/mL纳米钽处理组促增殖作用明显,可检测到明显的自噬;自噬抑制剂可明显抑制纳米钽处理细胞的增殖。结论纳米钽可通过诱导细胞自噬发挥促增殖作用,微米钽可能通过其他非自噬途径促进成骨细胞增殖。
Objective To investigate the effect of tantalum particles on the proliferation of osteoblasts andexplore its mechanism. Methods Mouse osteoblasts MC3T3-E1 were co-cultured with micro-tantalum particles(micro-Ta)and Nano-tantalum particles(nano-Ta)of different concentrations respectively. CCK-8 assay was usedto measure the cell viability at 6,12,24 and 48h. According to the result of CCK-8 the group with the most prolif-erative effect was screened and the level of autophagy was detected by using Western blot,laser confocal microsco-py and transmission electron microscopy(SEM). Finally,to verify the role of autophagy in pro-proliferation effectof nano-Ta,the OD value was measured repeatedly in combination with autophagy inducer and inhibitor. Results 100ng/m L micro-Ta treated groups had obvious proliferative effect but autophagy was not detected. 20μg/mL nano-Ta treated groups had obvious proliferative effect and autophagy was detected. CCK-8 assay revealed that autophagyinhibitor can significantly inhibited cell proliferation of nano-Ta treated group. Conclusion Nano-Ta could pro-mote cell proliferation by inducing autophagy,and micro-Ta may promote osteoblast proliferation through other non-autophagy pathway.
Objective To investigate the effect of tantalum particles on the proliferation of osteoblasts andexplore its mechanism. Methods Mouse osteoblasts MC3T3-E1 were co-cultured with micro-tantalum particles(micro-Ta)and Nano-tantalum particles(nano-Ta)of different concentrations respectively. CCK-8 assay was usedto measure the cell viability at 6,12,24 and 48h. According to the result of CCK-8 the group with the most prolif-erative effect was screened and the level of autophagy was detected by using Western blot,laser confocal microsco-py and transmission electron microscopy(SEM). Finally,to verify the role of autophagy in pro-proliferation effectof nano-Ta,the OD value was measured repeatedly in combination with autophagy inducer and inhibitor. Results 100ng/m L micro-Ta treated groups had obvious proliferative effect but autophagy was not detected. 20μg/mL nano-Ta treated groups had obvious proliferative effect and autophagy was detected. CCK-8 assay revealed that autophagyinhibitor can significantly inhibited cell proliferation of nano-Ta treated group. Conclusion Nano-Ta could pro-mote cell proliferation by inducing autophagy,and micro-Ta may promote osteoblast proliferation through other non-autophagy pathway.
引文
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[21]卢金婧.多发性硬化患者颅内深部灰质核团MRI的T2信号的定量分析及其临床意义[J].实用药物与临床,2017,20(4):412-415.
[22]王丹,孙刚,王瑶.蒲黄总黄酮含药血清对巨噬细胞自体吞噬及Akt/m TOR信号通路的影响[J].实用医学杂志,2017,33(16):2653-2657.
[23]蒋晶晶,姚鹏,田悦,等.NRG1-Erb B2信号通路对骨癌痛大鼠脊髓胶质细胞及IL-1β的影响[J].实用药物与临床,2016,19(6):657-660.
[2]TRAN P A,FOX K,TRAN N.Novel hierarchical tantalum oxide-PDMS hybrid coating for medical implants:One pot synthesis,characterization and modulation of fibroblast proliferation[J].JColloid Interface Sci,2017,485:106-115.
[3]FRANDSEN C J,BRAMMER K S,NOH K,et al.Tantalumcoating on Ti O2 nanotubes induces superior rate of matrix miner-alization and osteofunctionality in human osteoblasts[J].MaterSci Eng C Mater Biol Appl,2014,37:332-341.
[4]WANG L,HU X,MA X,et al.Promotion of osteointegration un-der diabetic conditions by tantalum coating-based surface modifi-cation on 3-dimensional printed porous titanium implants[J].Colloids Surf B Biointerfaces,2016,148:440-452.
[5]YANG J Y,PARK M Y,PARK S Y,et al.Nitric oxide-inducedautophagy in MC3T3-E1 cells is associated with cytoprotectionvia AMPK activation[J].Korean J Physiol Pharmacol,2015,19(6):507-514.
[6]WANG X,FENG Z,LI J,et al.High glucose induces autopha-gy of MC3T3-E1 cells via ROS-AKT-m TOR axis[J].Mol CellEndocrinol,2016,429:62-72.
[7]CONTRERAS R G,VILCHIS J R,SAKAGAMI H,et al.Typeof cell death induced by seven metals in cultured mouse osteo-blastic cells[J].In Vivo,2010,24(4):507-512.
[8]SCHLEE M,VAN DER SCHOOR W P,VAN DER SCHOOR AR.Immediate loading of trabecular metal-enhanced titanium den-tal implants:interim results from an international proof-of-princi-ple study[J].Clin Implant Dent Relat Res,2015,17(Suppl 1):e308-320.
[9]SCHLEE M,PRADIES G,MEHMKE W U,et al.Prospective,multicenter evaluation of trabecular metal-enhanced titanium dental implants placed in routine dental practices:1-year inter-im report from the development period(2010 to 2011)[J].ClinImplant Dent Relat Res,2015,17(6):1141-1153.
[10]ZHU Y,GU Y,QIAO S,et al.Bacterial and mammalian cellsadhesion to tantalum-decorated micro-/nano-structured titanium[J].J Biomed Mater Res A,2017,105(3):871-888.
[11]BONUTTI P M,PIVEC R,ISSA K,et al.Delamination of tanta-lum porous coating from a TKA due to regional dissemination ofdebris[J].Orthopedics,2013,36(8):600-604.
[12]ZHANG D,WONG C S,WEN C,et al.Cellular responses of os-teoblast-like cells to 17 elemental metals[J].J Biomed MaterRes A,2017,105(1):148-158.
[13]NINOMIYA J T,STRUVE J A,KROLIKOWSKI J,et al.Po-rous ongrowth surfaces alter osteoblast maturation and mineraliza-tion[J].J Biomed Mater Res A,2015,103(1):276-281.
[14]BALLA V K,BODHAK S,BOSE S,et al.Porous tantalumstructures for bone implants:fabrication,mechanical and in vi-tro biological properties[J].Acta Biomater,2010,6(8):3349-3359.
[15]SAKAI T,TAKEDA S,NAKAMURA M.The effects of particu-late metals on cell viability of osteoblast-like cells in vitro[J].Dent Mater J,2002,21(2):133-146.
[16]HUO W T,ZHAO L Z,YU S,et al.Significantly enhanced os-teoblast response to nano-grained pure tantalum[J].Sci Rep,2017,7:40868.
[17]KALUDEROVIC M R,MOJIC M,SCHRECKENBACH J P,etal.A key role of autophagy in osteoblast differentiation on titani-um-based dental implants[J].Cells Tissues Organs,2014,200(3-4):265-277.
[18]YANG L,MENG H,YANG M.Autophagy protects osteoblastsfrom advanced glycation end products-induced apoptosis throughintracellular reactive oxygen species[J].J Mol Endocrinol,2016,56(4):291-300.
[19]PEYNSHAERT K,MANSHIAN B B,JORIS F,et al.Exploitingintrinsic nanoparticle toxicity:the pros and cons of nanoparticle-induced autophagy in biomedical research[J].Chem Rev,2014,114(15):7581-7609.
[20]HA S W,WEITZMANN M N,BECK G R J R.Bioactive silicananoparticles promote osteoblast differentiation through stimula-tion of autophagy and direct association with LC3 and p62[J].ACS Nano,2014,8(6):5898-5910.
[21]卢金婧.多发性硬化患者颅内深部灰质核团MRI的T2信号的定量分析及其临床意义[J].实用药物与临床,2017,20(4):412-415.
[22]王丹,孙刚,王瑶.蒲黄总黄酮含药血清对巨噬细胞自体吞噬及Akt/m TOR信号通路的影响[J].实用医学杂志,2017,33(16):2653-2657.
[23]蒋晶晶,姚鹏,田悦,等.NRG1-Erb B2信号通路对骨癌痛大鼠脊髓胶质细胞及IL-1β的影响[J].实用药物与临床,2016,19(6):657-660.