氟化钠对成骨细胞MC3T3-E1分化及Wnt信号转导通路的影响
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摘要
目的探究氟化钠(NaF)对成骨细胞MC3T3-E1分化及Wnt信号转导通路的影响。方法小鼠成骨细胞MC3T3-E1细胞分为实验组和对照组,实验组细胞以终浓度为0. 05,0. 10,0. 50,1. 00,2. 00,3. 00,5. 00 mmol·L~(-1)NaF处理,对照组细胞则以等量生理盐水处理。用细胞计数试剂盒(CCK-8)法检测MC3T3-E1细胞增殖情况;用碱性磷酸酶(ALP)试剂盒检测MC3T3-E1细胞内ALP含量;蛋白免疫印迹法检测MC3T3-E1细胞Wnt信号转导通路蛋白的表达。结果 0. 05,0. 10,0. 50,1. 00,2. 00,3. 00,5. 00 mmol·L~(-1)NaF处理48 h后,MC3T3-E1细胞生存率分别为(99. 18±2. 15)%,(92. 64±3. 15)%,(83. 69±2. 04)%,(70. 16±2. 49)%,(56. 15±3. 57)%,(42. 36±3. 58)%,(29. 56±1. 36)%。干预24 h后,对照组和低、中、高剂量实验组(0. 50,2. 00,5. 00 mmol·L~(-1)NaF) MC3T3-E1细胞ALP含量分别为(10. 98±3. 52),(13. 26±2. 96),(8. 63±2. 05),(7. 12±1. 24) U·g-1prot,β-catenin蛋白相对表达量分别为0. 88±0. 02,1. 23±0. 06,0. 68±0. 05,0. 35±0. 05,Lrp5蛋白相对表达量分别为0. 96±0. 04,1. 32±0. 02,0. 65±0. 06,0. 33±0. 06,差异均有统计学意义(均P <0. 05)。结论高浓度NaF可抑制MC3T3-E1细胞增殖、分化,但低浓度的NaF却可促进MC3T3-E1细胞分化,其作用机制与调控Wnt信号转导通路蛋白表达相关。
Objective To explore the effect of sodium fluoride( NaF)on the differentiation and the Wnt signal transduction pathway in osteoblast MC3T3-E1. Methods Mouse osteoblast cells( MC3T3-E1)were divided into experiment group and control group, respectively,while the cells in experiment group were treated with 0. 05,0. 10,0. 50,1. 00,2. 00,3. 00,5. 00 mmol·L~(-1) of NaF,respectively,while the cells in control group were treated with equal amount of normal saline.The MC3T3-E1 cells proliferation were detected by cell counting kits-8( CCK-8) method; The activity of alkaline phosphatase( ALP) in MC3T3-E1 cells was detected with ALP kits,and the proteins expression of Wnt signal transduction pathway in MC3T3-E1 cells was detected by Western Blot. Results At 48 h after treatment( 0. 05,0. 10,0. 50,0. 00,1. 00,2. 00,3. 00 and 5. 00 mmol·L~(-1) of NaF),the survival rates were( 99. 18 ± 2. 15) %,( 92. 64 ± 3. 15) %,( 83. 69 ± 2. 04) %,( 70. 16 ± 2. 49) %,( 56. 15 ± 3. 57) %,( 42. 36 ± 3. 58) %,( 29. 56 ± 1. 36) %,respectively. After a 24-hour treatment,the ALP content of MC3T3-E1 cells in control group and experiment-L/-M/-H group( 0. 50,2. 00 mmol·L~(-1) NaF) were( 10. 98 ± 3. 52),( 13. 26 ± 2. 96),( 8. 63 ± 2. 05),( 7. 12 ± 1. 24) U·g-1 prot; the relative expression levels of β-catenin protein were 0. 88 ± 0. 02,1. 23 ± 0. 06,0. 68 ± 0. 05,0. 35 ± 0. 05; the relative expression levels of Lrp5 protein were 0. 96 ± 0. 04,1. 32 ± 0. 02,0. 65 ± 0. 06,0. 33 ± 0. 06,and there were significant differences among groups( P < 0. 05). Conclusion High concentration of NaF can inhibit the proliferation and differentiation of MC3T3-E1 cells,while the low concentration of NaF can promote the differentiation of MC3T3-E1 cells,and the mechanism might be associated with the regulation of Wnt signaling by protein-protein interaction.
Objective To explore the effect of sodium fluoride( NaF)on the differentiation and the Wnt signal transduction pathway in osteoblast MC3T3-E1. Methods Mouse osteoblast cells( MC3T3-E1)were divided into experiment group and control group, respectively,while the cells in experiment group were treated with 0. 05,0. 10,0. 50,1. 00,2. 00,3. 00,5. 00 mmol·L~(-1) of NaF,respectively,while the cells in control group were treated with equal amount of normal saline.The MC3T3-E1 cells proliferation were detected by cell counting kits-8( CCK-8) method; The activity of alkaline phosphatase( ALP) in MC3T3-E1 cells was detected with ALP kits,and the proteins expression of Wnt signal transduction pathway in MC3T3-E1 cells was detected by Western Blot. Results At 48 h after treatment( 0. 05,0. 10,0. 50,0. 00,1. 00,2. 00,3. 00 and 5. 00 mmol·L~(-1) of NaF),the survival rates were( 99. 18 ± 2. 15) %,( 92. 64 ± 3. 15) %,( 83. 69 ± 2. 04) %,( 70. 16 ± 2. 49) %,( 56. 15 ± 3. 57) %,( 42. 36 ± 3. 58) %,( 29. 56 ± 1. 36) %,respectively. After a 24-hour treatment,the ALP content of MC3T3-E1 cells in control group and experiment-L/-M/-H group( 0. 50,2. 00 mmol·L~(-1) NaF) were( 10. 98 ± 3. 52),( 13. 26 ± 2. 96),( 8. 63 ± 2. 05),( 7. 12 ± 1. 24) U·g-1 prot; the relative expression levels of β-catenin protein were 0. 88 ± 0. 02,1. 23 ± 0. 06,0. 68 ± 0. 05,0. 35 ± 0. 05; the relative expression levels of Lrp5 protein were 0. 96 ± 0. 04,1. 32 ± 0. 02,0. 65 ± 0. 06,0. 33 ± 0. 06,and there were significant differences among groups( P < 0. 05). Conclusion High concentration of NaF can inhibit the proliferation and differentiation of MC3T3-E1 cells,while the low concentration of NaF can promote the differentiation of MC3T3-E1 cells,and the mechanism might be associated with the regulation of Wnt signaling by protein-protein interaction.
引文
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[2]郑文伟,马玉环,陈后煌,等.跳骨片抑制大鼠膝骨关节炎Wnt/β-catenin信号转导通路介导炎症反应的机制研究[J].国际骨科学杂志,2017,38(1):60-64.
[3]刘媛,周道成,赵恒侠,等.基于Wnt/β-catenin通路探讨中医药防治糖尿病骨质疏松症[J].广州中医药大学学报,2017,34(4):604-608.
[4]李忠海,韩丽伟,赵彦涛,等.不同浓度锶对MC3T3-E1细胞增殖、ALP活性及成骨分化的影响[J].中国骨与关节杂志,2016,5(3):221-225.
[5]赵彦涛,李忠海,胡先同,等.不同浓度rhBMP-2对MC3T3-E1细胞增殖、ALP活性及成骨分化的影响[J].中国骨与关节损伤杂志,2016,31(3):281-284.
[6]MIAO D,ZHOU B,ZHOU X,et al. Thiazolidinediones inhibit mouse osteoblastic MC3T3-E1 cell proliferation in part through the Wnt signaling pathway[J]. J Inv Med,2015,63(5):758-762.
[7]LI W,JIANG B,CAO X,et al. Protective effect of lycopene on fluoride-induced ameloblasts apoptosis and dental fluorosis through oxidative stress-mediated Caspase pathways.[J]. Chem Biol Interact,2016,261(21):27-34.
[8]徐伟丽,牛玲玲,王文侠,等.经典Wnt信号通路对骨代谢的调节作用[J].中国骨质疏松杂志,2016,22(3):376-380.