金枪鱼鱼骨活性钙对鼠胚胎成骨细胞前体细胞(MC3T3-E1)活力和凋亡作用
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摘要
目的:以金枪鱼骨为原料,制备柠檬酸-苹果酸钙剂(CMC),研究其对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的细胞活力、凋亡的影响,并探讨其作用机制。方法:在小鼠胚胎成骨细胞前体细胞(MC3T3-E1)培养液中,分别加入高浓度(1μg/m L)CMC和低浓度(0.1μg/m L)CMC,利用MTT法检测对MC3T3-E1细胞活力的影响,流式细胞仪检测血清饥饿诱导的细胞凋亡。结果:高浓度和低浓度的钙对MC3T3-E1细胞活力均有显著的促进作用,24 h的高钙组的细胞增长率(9.67%)优于低钙组(8.95%);36 h和48 h后低钙组的增长率分别为14.96%和20.33%,明显优于36 h和48 h作用后的高钙组(6.23%和1.73%),两组细胞凋亡率与空白组比较差异较小,且均显著低于对照组(p<0.01)。结论:CMC高钙组和CMC低钙组在一定浓度范围内,能促进MC3T3-E1细胞活力显著增强,并且改善血清饥饿诱导的成骨前体细胞凋亡。
Objective: The aim of this study was to inspect the effect of citric-malic acid calcium( CMC) on the cell viability and apoptosis of mouse embryonic osteoblast precursor cells( MC3 T3-E1) was investigated by using tuna bone as raw material,and to explore the mechanism of action between CMC and cells. Methods: A high concentration( 1 μg/m L) of CMC and a low concentration( 0.1 μg/m L) of CMC were added to the culture medium of mouse embryonic osteoblast precursor cells( MC3 T3-E1),MTT assay was used to detect MC3 T3-E1 cell viability,flow cytometry was used to detect serum starvationinduced apoptosis. Results: It showed that high concentration and low concentration of calcium had a significant effect on the viability of MC3 T3-E1 cells.After incubation for 24 hours,the cell rate( 9.67%) in the high calcium group was better than that in the low calcium group( 8.95%). The proliferation rates of low calcium group were 14.96% and 20.33% at 36 h and 48 h,respectively,which were significantly higher than those in high calcium group( 6.23% and 1.73%) after 36 h and 48 h. The apoptotic rate of the two groups was significantly lower than that of the blank group( p < 0.01).Conclusion: CMC high calcium group and CMC low calcium group in a certain concentration range,can promote MC3 T3-E1 cell viability significantly enhanced,and inhibit the serum starvation-induced osteogenic precursor cell apoptosis.
Objective: The aim of this study was to inspect the effect of citric-malic acid calcium( CMC) on the cell viability and apoptosis of mouse embryonic osteoblast precursor cells( MC3 T3-E1) was investigated by using tuna bone as raw material,and to explore the mechanism of action between CMC and cells. Methods: A high concentration( 1 μg/m L) of CMC and a low concentration( 0.1 μg/m L) of CMC were added to the culture medium of mouse embryonic osteoblast precursor cells( MC3 T3-E1),MTT assay was used to detect MC3 T3-E1 cell viability,flow cytometry was used to detect serum starvationinduced apoptosis. Results: It showed that high concentration and low concentration of calcium had a significant effect on the viability of MC3 T3-E1 cells.After incubation for 24 hours,the cell rate( 9.67%) in the high calcium group was better than that in the low calcium group( 8.95%). The proliferation rates of low calcium group were 14.96% and 20.33% at 36 h and 48 h,respectively,which were significantly higher than those in high calcium group( 6.23% and 1.73%) after 36 h and 48 h. The apoptotic rate of the two groups was significantly lower than that of the blank group( p < 0.01).Conclusion: CMC high calcium group and CMC low calcium group in a certain concentration range,can promote MC3 T3-E1 cell viability significantly enhanced,and inhibit the serum starvation-induced osteogenic precursor cell apoptosis.
引文
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[12]王凯,魏博,谭佳妮,等.珍珠粉对MC3T3-E1细胞增殖、分化、矿化及骨相关基因表达的影响[J].中国骨质疏松杂志,2014,20(6):666-672.
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[15]祁云云,张凯,丁晓墅,等.国内活性钙研究现状[J].天津化工,2006,20(2):14-15.
[16]马海霞,杨贤庆,李来好,等.微生物发酵罗非鱼骨粉工艺条件的优化[J].食品科学,2013,34(3):193-197.
[17]杨泽琴.钒钼酸铵分光光度示差法测定过磷酸钙中有效磷[J].化学工业与工程技术,1995,16(4):52-54.
[18]王鸿飞,飞刘,超徐,等.费菜总黄酮调节血脂及对肝癌细胞增殖的作用[J].中国食品学报,2013,13(4):23-26.
[19]Manolagas S C,Jilka R L.Bone marrow,cytokines,and bone remodeling.Emerging insights into the pathophysiology of osteoporosis[J].The New England journal of medicine,1995,332(5):305-11.
[20]Owen TA,Aronow M,Shalhoub V,et al.Progressive development of the rat osteoblast phenotype in vitro:Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix[J].Journal of Cellular physiology,1990,143(3):420-430.
[21]Tsai J A,Bucht E,Torring O,et al.Extracellular calcium increases free cytoplasmic calcium and DNA synthesis in human osteoblasts.[J].Hormone and Metabolic Research,2004,36(1):22-6.
[22]唐海林,苏琦.钙离子信号与细胞凋亡关系的研究进展[J].南华大学学报,2006,34(5):663-666.
[23]安洋,张慧宇,郭俊峰,等.降钙素基因相关肽对血清饥饿作用下MC3T3-E1成骨细胞凋亡和自噬的影响[J].华西口腔医学杂志,2017,35(2):133-138.
[24]Igney FH,Krammer PH.Death and anti-death:tumour resistance to apoptosis[J].Nature Reviews Cancer,2002,2(4):277-88.
[2]贾建萍,周彦钢,林赛君,等.金枪鱼骨营养成分分析[J].食品工业科技,2013,34(10):334-337.
[3]方健民,黄富雄,郑钟新,等.金枪鱼的营养价值和加工利用[J].水产科技,2006,23(2):8-13.
[4]王霞,李妍妍,王奇,等.金枪鱼胰脏酶解液对糖尿病大鼠血糖和血脂的作用[J].中国食品学报,2012,12(7):24-28.
[5]王霞,黄健,李妍妍,等.金枪鱼鱼油对D-半乳糖致衰老小鼠学习记忆的作用[J].中国食品学报,2012,12(10):24-28.
[6]王珊珊,李八方,周德庆,等.鳕鱼骨活性钙的正交实验优化及其生物利用度分析[J].食品科学,2015,36(20):13-18.
[7]易美华,杨仕生,谢福美.罗非鱼骨制备活性钙的技术研究[J].食品研究与开发,2008,29(12):85-88.
[8]范鸿冰,汪之颖,刘鹏,等.鲢鱼骨胶原多肽螯合钙的制备研究[J].南方水产科学,2014,10(2):72-79.
[9]邵明栓,陶敏,向蔚,等.斑点叉尾鮰鱼脱脂及其制备CMC活性钙的工艺优化[J].食品科学,2010,31(20):111-115.
[10]Nemati Mahnaz,Kamilah Hanisah,Huda Nurul,et al.In vitro calcium availability in bakery products fortified with tuna bone powder as a natural calcium source.[J].International Journal of Food Sciences and Nutrition,2015,67(5):535-40.
[11]卓丽玲,张春岭,顾建红,等.钙对体外培养大鼠成骨细胞增殖分化及细胞周期的影响[J].中国兽医学报,2009,29(6):769-787.
[12]王凯,魏博,谭佳妮,等.珍珠粉对MC3T3-E1细胞增殖、分化、矿化及骨相关基因表达的影响[J].中国骨质疏松杂志,2014,20(6):666-672.
[13]芦红艳,李妍妍,王奇,等.鮟鱇鱼骨对去卵巢骨质疏松大鼠的影响[J].食品科学,2012,33(3):252-255.
[14]霍健聪,邓尚贵,童国忠.鳕鱼骨钙片的制备及其生物利用[J].水产学报,2010,34(3):382-388.
[15]祁云云,张凯,丁晓墅,等.国内活性钙研究现状[J].天津化工,2006,20(2):14-15.
[16]马海霞,杨贤庆,李来好,等.微生物发酵罗非鱼骨粉工艺条件的优化[J].食品科学,2013,34(3):193-197.
[17]杨泽琴.钒钼酸铵分光光度示差法测定过磷酸钙中有效磷[J].化学工业与工程技术,1995,16(4):52-54.
[18]王鸿飞,飞刘,超徐,等.费菜总黄酮调节血脂及对肝癌细胞增殖的作用[J].中国食品学报,2013,13(4):23-26.
[19]Manolagas S C,Jilka R L.Bone marrow,cytokines,and bone remodeling.Emerging insights into the pathophysiology of osteoporosis[J].The New England journal of medicine,1995,332(5):305-11.
[20]Owen TA,Aronow M,Shalhoub V,et al.Progressive development of the rat osteoblast phenotype in vitro:Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix[J].Journal of Cellular physiology,1990,143(3):420-430.
[21]Tsai J A,Bucht E,Torring O,et al.Extracellular calcium increases free cytoplasmic calcium and DNA synthesis in human osteoblasts.[J].Hormone and Metabolic Research,2004,36(1):22-6.
[22]唐海林,苏琦.钙离子信号与细胞凋亡关系的研究进展[J].南华大学学报,2006,34(5):663-666.
[23]安洋,张慧宇,郭俊峰,等.降钙素基因相关肽对血清饥饿作用下MC3T3-E1成骨细胞凋亡和自噬的影响[J].华西口腔医学杂志,2017,35(2):133-138.
[24]Igney FH,Krammer PH.Death and anti-death:tumour resistance to apoptosis[J].Nature Reviews Cancer,2002,2(4):277-88.