低水平镉暴露对破骨细胞分化的影响
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摘要
为研究低水平镉(Cd)暴露对破骨细胞(osteoclast,OC)分化的影响,试验以RAW264.7细胞(单核巨噬细胞系)为材料,在巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)存在的条件下,用不同浓度Cd处理4 d;利用CCK-8法检测破骨细胞及其前体细胞活性变化,抗酒石酸酸性磷酸酶(TRAP)染色试验观察破骨细胞生成,激光共聚焦显微镜观察破骨细胞形态变化,蛋白免疫印迹(Western blot)技术和荧光定量聚合酶链式反应(qRT-PCR)检测破骨细胞标志性蛋白及其mRNA水平。结果显示,随着Cd浓度升高,细胞活力受到明显的抑制(P<0.01),并呈浓度-效应关系;与对照组相比,破骨细胞产生的数目和面积均显著或极显著下降(P<0.05或P<0.01);2、5μmol·L~(-1) Cd处理组破骨细胞封闭带的形成均受到抑制;2和5μmol·L~(-1) Cd处理组破骨细胞特异性蛋白及其mRNA表达量均显著或极显著下降(P<0.05或P<0.01),并呈剂量依赖性。结果表明,低微摩尔水平镉暴露能够抑制破骨细胞的分化。
In order to study the effect of low level cadmium(Cd) exposure on osteoclast(OC) differentiation, RAW264.7 cell(mononuclear macrophage lineage) was used as materials. In the presence of macrophage colony stimulating factor(M-CSF) and receptor-activated nuclear factor κB ligand(RANKL), treatment of different concentrations of Cd were conducted for 4 days. CCK-8 assay was used to detect changes in the viability of osteoclasts and their precursor cells. Tartrate-resistant acid phosphatase(TRAP) staining assay was used to observe the osteoclastogenesis. Laser scanning confocal microscopy was used to observe morphological changes of osteoclasts. The expression of osteoclast marker proteins and mRNA expression were detected by Western blot and qRT-PCR. The results showed that with the increase of Cd concentration, cell viability was significantly inhibited(P<0.01),which showed a concentration-effect relationship. Compared with the control group, the number and area of osteoclast production were significantly or extremely significantly decreased(P<0.05 or P<0.01). In the 2 and 5 μmol·L~(-1) Cd treatment groups, the formation of osteoclasts' sealing zone was inhibited. The osteoclast-specific protein and mRNA expression levels of 2 and 5 μmol·L~(-1) Cd groups were significantly or extremely significantly decreased(P<0.05 or P<0.01), which showed the dose effect. The above results indicate that low micromolar cadmium exposure can inhibit the differentiation of osteoclasts.
In order to study the effect of low level cadmium(Cd) exposure on osteoclast(OC) differentiation, RAW264.7 cell(mononuclear macrophage lineage) was used as materials. In the presence of macrophage colony stimulating factor(M-CSF) and receptor-activated nuclear factor κB ligand(RANKL), treatment of different concentrations of Cd were conducted for 4 days. CCK-8 assay was used to detect changes in the viability of osteoclasts and their precursor cells. Tartrate-resistant acid phosphatase(TRAP) staining assay was used to observe the osteoclastogenesis. Laser scanning confocal microscopy was used to observe morphological changes of osteoclasts. The expression of osteoclast marker proteins and mRNA expression were detected by Western blot and qRT-PCR. The results showed that with the increase of Cd concentration, cell viability was significantly inhibited(P<0.01),which showed a concentration-effect relationship. Compared with the control group, the number and area of osteoclast production were significantly or extremely significantly decreased(P<0.05 or P<0.01). In the 2 and 5 μmol·L~(-1) Cd treatment groups, the formation of osteoclasts' sealing zone was inhibited. The osteoclast-specific protein and mRNA expression levels of 2 and 5 μmol·L~(-1) Cd groups were significantly or extremely significantly decreased(P<0.05 or P<0.01), which showed the dose effect. The above results indicate that low micromolar cadmium exposure can inhibit the differentiation of osteoclasts.
引文
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[13] LIM H S, LEE H H, KIM T H, et al. Relationship between heavy metal exposure and bone mineral density in korean adult[J]. J Bone Metab, 2016, 23(4):223-231.
[14] 宋瑞龙. OPG对破骨细胞分化过程中细胞骨架的影响及其分子机理[D]. 扬州:扬州大学, 2014.SONG R L. Influences of osteoprotegerin on the cytoskeleton of osteoclast and the involve signalling pathways in the process of osteoclast differentiation[D]. Yangzhou:Yangzhou University, 2014. (in Chinese)
[15] BRZóSKA M M, MONIUSZKO-JAKONIUK J. Low-level lifetime exposure to cadmium decreases skeletal mineralization and enhances bone loss in aged rats[J]. Bone, 2004, 35(5):1180-1191.
[16] ENGSTR?M A, MICHA, et al. Long-term cadmium exposure and the association with bone mineral density and fractures in a population-based study among women[J]. J Bone Miner Res, 2011, 26(3):486-495.
[17] LIU W, DAI N N, WANG Y, et al. Role of autophagy in cadmium-induced apoptosis of primary rat osteoblasts[J]. Sci Rep, 2016, 6:20404.
[18] 赵鸿雁. 骨保护素对破骨细胞及其前体黏附结构和融合的影响[D]. 扬州:扬州大学, 2015.ZHAO H Y. Effects of osteoprotegerin on the peripheral adhesive structures and fusion of (pre)osteoclast[D]. Yangzhou:Yangzhou University, 2015. (in Chinese)
[19] TAKITO J, INOUE S, NAKAMURA M. The sealing zone in osteoclasts:a self-organized structure on the bone[J]. Int J Mol Sci, 2018, 19(4):984.
[20] BATSIR S, GEIGER B, KAM Z. Dynamics of the sealing zone in cultured osteoclasts[J]. Cytoskeleton (Hoboken), 2017, 74(2):72-81.
[2] WALLIN M, BARREGARD L, SALLSTEN G, et al. Response to “low-level cadmium exposure and bone health”[J]. J Bone Miner Res, 2017, 32(2):420-421.
[3] SINGHANIA S, SINGH S, PARIKH P. Cadmium as a risk factor for osteoporosis in COPD[J]. Int J Tuberc Lung Dis, 2017, 21(2):244.
[4] SCIMECA M, FEOLA M, ROMANO L, et al. Heavy metals accumulation affects bone microarchitecture in osteoporotic patients[J]. Environ Toxicol, 2017, 32(4):1333-1342.
[5] WEAVER V M, KIM N S, JAAR B G, et al. Associations of low-level urine cadmium with kidney function in lead workers[J]. Occup Environ Med, 2011, 68(4):250-256.
[6] CHEN X, ZHU G Y, GU S Z, et al. Effects of cadmium on osteoblasts and osteoclasts in vitro[J]. Environ Toxicol Pharmacol, 2009, 28(2):232-236.
[7] LIU W, ZHAO H Y, WANG Y, et al. Calcium-calmodulin signaling elicits mitochondrial dysfunction and the release of cytochrome c during cadmium-induced apoptosis in primary osteoblasts[J]. Toxicol Lett, 2014, 224(1):1-6.
[8] V??N?NEN H K, LAITALA-LEINONEN T. Osteoclast lineage and function[J]. Arch Biochem Biophys, 2008, 473(2):132-138.
[9] WALLIN M, BARREGARD L, SALLSTEN G, et al. Low-level cadmium exposure is associated with decreased bone mineral density and increased risk of incident fractures in elderly men:the MrOS sweden study[J]. J Bone Miner Res, 2016, 31(4):732-741.
[10] AKESSON A, BJELLERUP P, LUNDH T, et al. Cadmium-induced effects on bone in a population-based study of women[J]. Environ Health Perspect, 2006, 114(6):830-834.
[11] CHEN X, GAN C H, ZHU G Y, et al. Benchmark dose for estimation of cadmium reference level for osteoporosis in a Chinese female population[J]. Food Chem Toxicol, 2013, 55:592-595.
[12] TAHA M M, ABDALLAH H, SHAHY E M, et al. Impact of occupational cadmium exposure on bone in sewage workers [J]. Int J Occup Environ Health, 2018, 24(3-4):101-108.
[13] LIM H S, LEE H H, KIM T H, et al. Relationship between heavy metal exposure and bone mineral density in korean adult[J]. J Bone Metab, 2016, 23(4):223-231.
[14] 宋瑞龙. OPG对破骨细胞分化过程中细胞骨架的影响及其分子机理[D]. 扬州:扬州大学, 2014.SONG R L. Influences of osteoprotegerin on the cytoskeleton of osteoclast and the involve signalling pathways in the process of osteoclast differentiation[D]. Yangzhou:Yangzhou University, 2014. (in Chinese)
[15] BRZóSKA M M, MONIUSZKO-JAKONIUK J. Low-level lifetime exposure to cadmium decreases skeletal mineralization and enhances bone loss in aged rats[J]. Bone, 2004, 35(5):1180-1191.
[16] ENGSTR?M A, MICHA, et al. Long-term cadmium exposure and the association with bone mineral density and fractures in a population-based study among women[J]. J Bone Miner Res, 2011, 26(3):486-495.
[17] LIU W, DAI N N, WANG Y, et al. Role of autophagy in cadmium-induced apoptosis of primary rat osteoblasts[J]. Sci Rep, 2016, 6:20404.
[18] 赵鸿雁. 骨保护素对破骨细胞及其前体黏附结构和融合的影响[D]. 扬州:扬州大学, 2015.ZHAO H Y. Effects of osteoprotegerin on the peripheral adhesive structures and fusion of (pre)osteoclast[D]. Yangzhou:Yangzhou University, 2015. (in Chinese)
[19] TAKITO J, INOUE S, NAKAMURA M. The sealing zone in osteoclasts:a self-organized structure on the bone[J]. Int J Mol Sci, 2018, 19(4):984.
[20] BATSIR S, GEIGER B, KAM Z. Dynamics of the sealing zone in cultured osteoclasts[J]. Cytoskeleton (Hoboken), 2017, 74(2):72-81.