HEMA促进人牙源性间充质细胞自噬水平的作用研究

摘要/Abstract

摘要： 目的　研究树脂单体甲基丙烯酸羟乙酯（2-Hydroxyethyl methacrylate，HEMA）对人牙源性间充质细胞（dental mesenchymal cells，DMCs）的自噬水平的作用。方法从正畸或预防性拔除的新鲜健康年轻恒牙(15-22岁患者)牙髓中，获得体外牙髓间充质细胞。培养至4~6代后，以不同浓度的HEMA培养DMCs。CCK-8检测HEMA(0,2,4,6,8,10,12mmol/L)处理12、24、48 h细胞增殖的情况。鬼笔环肽（Phalloidin）检测细胞结构的变化。吖啶橙（Acridine Orange，AO）检测自噬酸性泡的形成。Western blot检测细胞中Beclin1、Atg5和LC3II的表达。结果随着HEMA浓度的增加，DMCs增殖明显受抑制，呈现时间-浓度依赖性。HEMA处理组细胞结构改变，并可见明显增加的自噬酸性泡聚集。与对照组相比，HEMA处理组中Beclin1、Atg5和LC3II的表达明显升高，且呈浓度依赖性。结论HEMA对人牙源性间充质细胞有明显的毒性作用，其机制与细胞自噬活化水平的升高有关。

关键词: HEMA, 人牙源性间充质细胞, 细胞毒性, 自噬

Abstract: Objective　 To investigate the autophagy-induction effect of 2-Hydroxyethyl methacrylate(HEMA) on human dental mesenchymal cells (DMCs). Methods　DMCs were obtained from freshly extracted healthy premolars and third molars from patients (15–22 years old) with orthodontic and preventive teeth extraction. Cells of the 4th-6th generations were treated by different concentrations of HEMA for different periods. Cell viability was detected by CCK-8 assessment after treated with HEMA (0,2,4,6,8,10,12 mmol/L) for 12, 24, 48 h. Cell phenotype change was detected by Phalloidin staining. AO staining was used to observe the vesicular organelles in DMCs. Western blot assessment was applied to detected the protein expressions of Beclin1, Atg5 and LC3II. Results　The cell viability decreased with HEMA treatment in a dose- and time-dependent manner. The cell phenotype was changed by HEMA treatment. Increasing numbers of vesicular organelles were observed in the cytoplasm of HEMA-treated DMCs. The protein expressions of Beclin1, Atg5 and LC3II were elevated by HEMA treatment in DMCs in dose-dependent manner. Conclusion　HEMA greatly enhances the autophagy induction in a dose –dependent manner, which contributes to the cytotoxicity in DMCs. Its mechanism is associated with the increase of cell autophagy activation levels.

Key words: human dental mesenchymal cells, cytotoxicity, autophagy

中图分类号:

R329.28

王维倩黄震李靖敏丁子清. HEMA促进人牙源性间充质细胞自噬水平的作用研究[J]. , 2018, 38(12): 1084-1088.

参考文献

[1]Tuncer S, Demirci M, Schweikl H, et al.Inhibition of cell survival,viability and proliferation by dentin adhesives after direct and indirect exposure in vitro[J].Clin Oral Investig, 2012, 16(6):1635-1646
[2]Schweikl H, Petzel C, Bolay C, et al.Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway[J].Biomaterials, 2014, 35(9):2890-2904
[3]Schweikl H, Spagnuolo G, Schmalz G.Genetic and Cellular Toxicology of Dental Resin Monomers[J].J Dent Res, 2006, 85(10):870-877
[4]Cetingü? A, Olmez S, Vural N.HEMA diffusion from dentin bonding agents in young and old primary molars in vitro[J].Dental Materials, 2007, 23(3):302-307
[5]Inamitsu H, Okamoto K, Sakai E, et al.The dental resin monomers HEMA and TEGDMA have inhibitory effects on osteoclast differentiation with low cytotoxicity[J].J Appl Toxicol, 2017, 37(7):817-824
[6]Ginzkey C, Zinnitsch S, Steussloff G, et al.Assessment of HEMA and TEGDMA induced DNA damage by multiple genotoxicological endpoints in human lymphocytes[J].Dent Mater, 2015, 31(8):865-876
[7]Schweikl H, Petzel C, Bolay C, et al.Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway[J].Biomaterials, 2014, 35(9):2890-2904
[8] Yin J, Ye A J, Tan K S.Autophagy is involved in starvation response and cell death in Blastocystis[J]. Microbiology, 2010, 156:665-677.
[9]Levine B.Autophagy in cell death: an innocent convict?[J].Journal of Clinical Investigation, 2005, 115(10):2679-2688
[10]Eddleston C L, Hindle A R, Agee K A, et al.Dimensional changes in acid-demineralized dentin matrices following the use of HEMA-water versus HEMA-alcohol primers[J].J Biomed Mater Res A, 2003, 67(3):900-907
[11]Durner J, Spahl W, Zaspel J, et al.Eluted substances from unpolymerized and polymerized dental restorative materials and their Nernst partition coefficient[J].Dent Mater, 2010, 26(1):91-99
[12]Eckhardt A, Muller P, Hiller K A, et al.Influence of TEGDMA on the mammalian cell cycle in comparison with chemotherapeutic agents[J].Dent Mater, 2010, 26(3):232-241
[13]Gallorini M, Cataldi A, di Giacomo V.HEMA-induced cytotoxicity: oxidative stress,genotoxicity and apoptosis[J].Int Endod J, 2014, 47(9):813-818
[14] Bolling A K, Solhaug A, Morisbak E, et al.The dental monomer hydroxyethyl methacrylate (HEMA) counteracts lipopolysaccharide-induced IL-1β release—Possible role of glutathione[J]. Toxicology Letters, 2017, 270:25-33.
[15]Schweikl H, Petzel C, Bolay C, et al.Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway[J].Biomaterials, 2014, 35(9):2890-2904
[16]Krifka S, Hiller K A, Spagnuolo G, et al.The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA)[J].Biomaterials, 2012, 33(21):5177-5186
[17]Chang HH, Guo MK, Kasten F H, et al.Stimulation of glutathione depletion,ROS production and cell cycle arrest of dental pulp cells and gingival epithelial cells by HEMA[J].Biomaterials, 2005, 26(7):745-753
[18]Bakopoulou A, Leyhausen G, Volk J, et al.Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stemprogenitor cells derived from deciduous teeth[J].Dental Materials, 2011, 27(6):608-617
[19]Andersson J, Dahlgren U.Effects on mouse immunity of long-term exposure in vivo to minute amounts of HEMA[J].Eur J Oral Sci, 2011, 119(2):109-114
[20] Saha S, Panigrahi D P, Patil S, et al.Autophagy in health and disease: A comprehensive review[J]. Biomed Pharmacother, 2018, 104:485-495.
[21]Mizushima N, Levine B, Cuervo AM, et al.Autophagy fights disease through cellular self-digestion[J].Nature, 2008, 451(7182):1069-1075
[22]Wang R C, Wei Y, An Z, et al.Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation[J].Science, 2012, 338(6109):956-959
[23]Mari?o G, Niso-Santano M, Baehrecke EH, et al.Self-consumption: the interplay of autophagy and apoptosis[J].Nat Rev Mol Cell Biol, 2014, 15(2):81-94
[24]Krolenko SA, Adamyan SY, Belyaeva TN, et al.Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres[J].Cell Biol Int, 2006, 30(11):933-939
[25]Yang J, Wan C, Nie S, et al.Localization of Beclin1 in mouse developing tooth germs: possible implication of the interrelation between autophagy and apoptosis[J].J Mol Histol, 2013, 44(6):619-627
[26]O' Sullivan TE, Geary CD, Weizman OE, et al.Atg5 Is Essential for the Development and Survival of Innate Lymphocytes[J].Cell Rep, 2016, 15(9):1910-1919
[27]Mukae K, Inoue Y, Moriyasu Y.ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells[J].Plant Signal Behav, 2015, 10(11):e1086859-
[28]Wang S, Li B, Qiao H, et al.Autophagy-related gene Atg5 is essential for astrocyte differentiation in the developing mouse cortex[J].EMBO Rep, 2014, 15(10):1053-1061
[29]Schaaf MB, Keulers TG, Vooijs MA, et al.LC3GABARAP family proteins: autophagy-(un)related functions[J].FASEB J, 2016, 30(12):3961-3978
[30]Yang A, Hacheney I, Wu YW.Semisynthesis of autophagy protein LC3 conjugates[J].Bioorg Med Chem, 2017, 25(18):4971-4976
[31]Sun Y, Liu MM, Lei XY, et al.SFTS phlebovirus promotes LC3-II accumulation and nonstructural protein of SFTS phlebovirus co-localizes with autophagy proteins[J].Sci Rep, 2018, 8(1):5287-