羊骨髓肽-钙螯合物对诱导分化的破骨细胞形成和吸收的影响
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摘要
目的探讨羊骨髓肽(sheep bone marrow peptides,MP)及其钙螯合物(MP-Ca)对诱导分化的OC形成和活性的影响。方法将MP与CaCl_2螯合制备MP-Ca,以RAW264.7为破骨前体细胞模型,建立OC的RANKL诱导体系,以MP和MP-Ca干预诱导过程,确定二者在最适作用浓度下对诱导曲线、TRAP活性和骨吸收陷窝数的影响。结果诱导后第5天,细胞呈现OC典型形态,TRAP染色呈阳性并在共培养的骨片上形成骨吸收陷窝,MTT检测时OD_(490)达到峰值;以第5天作为检测时间,MP与MP-Ca均以10~3μg/mL时OD_(490)最低;以10~3μg/mL MP和MP-Ca干预诱导体系,除第1天外,干预组OD_(490)均显著低于空白组(P<0.05);在第5天,MP-Ca组的OD_(490)和TRAP活性均显著低于MP组(P<0.05);各干预组的骨吸收陷窝数均显著低于空白组,且MP组显著低于MP-Ca组(P<0.05);17β-雌二醇(10μg/mL)的各检测指标均显著低于干预组(P<0.05)。结论虽效果不及雌激素,MP和MP-Ca均可抑制OC的诱导形成及其破骨活性,MP-Ca对OC诱导形成的抑制作用强于MP。
Objective Osteoclast numbers and activity are directly related to bone turnover and metabolism. This study aimed to investigate the effects of sheep bone marrow peptides(MP) and calcium-chelated MP(MP-Ca) on the formation and activity of RANKL-induced osteoclasts. Methods MP-Ca was prepared by a chelating reaction between MPs and CaCl_2. RAW264.7 cells were induced to osteoclasts by RANKL treatment, and cell proliferation was examined using the MTT assay. Induction was intervened by MP and MP-Ca to determine optimal concentrations and to investigate their effects on induction curves, TRAP activity, and the number of lacunae formed in bone slices. Results Under the induction system, on day 5, cultured cells were took on typical morphology of mature osteoclasts, TRAP staining was positive, resorptive pits formed, and the peak OD_(490 )value was achieved. The lowest OD_(490) value was achieved when 10~3 μg/mL MP or MP-Ca were added into the induction system on day 5. When 10~3 μg/mL MP or MP-Ca was added to the induction system, except on day 1, OD_(490) values from days 2-8 were significantly lower than the blank group(P< 0.05). In addition, the OD_(490) value and TRAP activity of the MP-Ca group were markedly lower than the MP group(P< 0.05). Pit numbers in the intervention groups were notably lower than the blank group, and the number of lacunae in the MP group was significantly lower than the MP-Ca group(P< 0.05). All indices checked in the 17-β-estradiol group were lower than the intervention groups(P<0.05). Conclusions Although inferior to estrogen, both MP and MP-Ca inhibite the RANKL-induced formation and the resorptive activity of osteoclasts. Moreover, MP-Ca was more effective at inhibiting osteoclast formation than MP.
Objective Osteoclast numbers and activity are directly related to bone turnover and metabolism. This study aimed to investigate the effects of sheep bone marrow peptides(MP) and calcium-chelated MP(MP-Ca) on the formation and activity of RANKL-induced osteoclasts. Methods MP-Ca was prepared by a chelating reaction between MPs and CaCl_2. RAW264.7 cells were induced to osteoclasts by RANKL treatment, and cell proliferation was examined using the MTT assay. Induction was intervened by MP and MP-Ca to determine optimal concentrations and to investigate their effects on induction curves, TRAP activity, and the number of lacunae formed in bone slices. Results Under the induction system, on day 5, cultured cells were took on typical morphology of mature osteoclasts, TRAP staining was positive, resorptive pits formed, and the peak OD_(490 )value was achieved. The lowest OD_(490) value was achieved when 10~3 μg/mL MP or MP-Ca were added into the induction system on day 5. When 10~3 μg/mL MP or MP-Ca was added to the induction system, except on day 1, OD_(490) values from days 2-8 were significantly lower than the blank group(P< 0.05). In addition, the OD_(490) value and TRAP activity of the MP-Ca group were markedly lower than the MP group(P< 0.05). Pit numbers in the intervention groups were notably lower than the blank group, and the number of lacunae in the MP group was significantly lower than the MP-Ca group(P< 0.05). All indices checked in the 17-β-estradiol group were lower than the intervention groups(P<0.05). Conclusions Although inferior to estrogen, both MP and MP-Ca inhibite the RANKL-induced formation and the resorptive activity of osteoclasts. Moreover, MP-Ca was more effective at inhibiting osteoclast formation than MP.
引文
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[13] 王珊珊. 鳕鱼骨胶原肽与活性钙的制备及其抗骨质疏松活性研究[D]. 中国海洋大学, 2013.Wang SS. Preparation and anti-osteoporotic activity of collagen peptide and active calcium of carp[D]. Ocean University of China, 2013.
[14] 吴庆儒. 雌激素和孕激素对体外培养奶牛破骨细胞增殖分化的影响[D]. 华中农业大学, 2007.Wu QR. Effects of estrogen and progesterone on the proliferation and differentiation of osteoclasts in dairy cows[D]. Huazhong Agricultural University, 2007.
[15] 付应霄, 顾建红, 赵鸿雁,等. 骨保护素对破骨细胞活性的影响[J]. 中国兽医学报, 2013, 33(5):738-741.Fu YX, Gu JH, Zhao HY, et al. Effect of osteoprotegerin on the activity of osteoclasts[J]. Chin J Vet Sci, 2013, 33(5):738-741.
[16] Kilic D, Kesim S, Liman N, et al. Comparison of the effects of dental filling Materials on mouse fibroblasts[J]. Biotechnol Biotechnol Eq, 2012, 26(4):3155-3162.
[17] Prause M, Seeliger C, Unger M, et al. Pantoprazole decreases cell viability and function of human osteoclasts in vitro[J]. Mediators Inflamm, 2015, 2015:413097.
[18] 李鹏辉, 田宗成, 商澎. 破骨细胞骨吸收功能的检测方法[J]. 生物学杂志, 2012, 29(4):74-77.Li PH, Tian ZC, Shang Y. Detection of bone resorption by osteoclasts[J]. J Biol, 2012, 29(4):74-77.
[2] 赵文韬, 张晓刚, 王志鹏. 自噬与骨质疏松症的相关性[J].中国生物化学与分子生物学报, 2017, 33(4):337- 341.Zhao WZ, Zhang XG, Wang ZP. Correlation between autophagy and osteoporosis[J]. Chin J Biochem Mol Biol, 2017, 33(4):337- 341.
[3] Bronsk■ J, Pr■sa R, Nevoral J. The role of amylin and related peptides in osteoporosis [J]. Clin Chim Acta, 2006, 373(1): 9-16.
[4] 金小岚. 钙剂和维生素D在骨质疏松症中的应用[J]. 中国临床医生杂志, 2013, 41(6):3-5.Jin XQ. Application of calcium and vitamin D in osteoporosis[J]. Chin Clin Dr, 2013, 41(6):3-5.
[5] 郭丹郡, 侯焘, 何慧. 生物活性肽促钙吸收机制与钙离子通道[J]. 食品安全质量检测学报, 2016, 7(11):4531-4535.Guo DJ, Hou X, He H. Bioactive peptides promote calcium absorption and calcium channels[J]. Food Safe Qual Detec Technol, 2016, 7(11):4531-4535.
[6] 程稚玲,霍乃蕊. 钙螯合胶原短肽的制备及其对去卵巢大鼠的骨质改善作用[D]. 山西农业大学, 2016.Cheng ZL, Huo NR. Preparation of calcium-chelating collagen peptides and their osteolytic effects on ovariectomized rats[D]. Shanxi Agricultural University, 2016
[7] 高文伟, 申勇涛, 程稚玲,等. 钙螯合胶原多肽与雌激素对去卵巢大鼠骨质改善作用的比较[J]. 中国实验动物学报, 2017, 25(3):256-262.Gao WW, Shen YT, Cheng ZL, et al. Comparison of calcium chelating collagen peptides and estrogen on osteoporosis in ovariectomized rats[J]. Acta Lab Anim Sci Sin, 2017, 25(3): 256-262.
[8] 甄守艳,霍乃蕊. 羊骨胶原肽的酶法制备及肽钙螯合研究[D]. 山西农业大学, 2015.Zhen SY, Huo NR. Enzymatic preparation and peptide chelated sheep bone collagen peptide research [D]. Shanxi Agricultural University, 2015.
[9] Guo H, Hong Z, Yi R. Core-Shell collagen peptide chelated calcium/calcium alginate nanoparticles from fish scales for calcium supplementation[J]. J Food Sci, 2015, 80(7):N1595-1601.
[10] Nakchum L, Kim SM. Preparation of squid skin collagen hydrolysate as an antihyaluronidase, antityrosinase, and antioxidant agent[J]. Prep Biochem Biotechnol, 2016, 46(2):123-130.
[11] 闫景刚, 段卫华. 骨质疏松症治疗药物的研究进展[J]. 中国医药导报, 2015, 12(9):34-37.Yan JG, Duan WH. Research progress in the treatment of osteoporosis[J]. Chin Med Herald, 2015, 12(9): 34-37.
[12] 许先猛, 董文宾. 猪皮胶原多肽螯合钙增加大鼠骨密度[J]. 食品科学, 2017(23):191-195.Xu XM, Dong WB. Increased bone mineral density in pigs with chelating collagen peptides[J]. Food Sci, 2017(23):191-195.
[13] 王珊珊. 鳕鱼骨胶原肽与活性钙的制备及其抗骨质疏松活性研究[D]. 中国海洋大学, 2013.Wang SS. Preparation and anti-osteoporotic activity of collagen peptide and active calcium of carp[D]. Ocean University of China, 2013.
[14] 吴庆儒. 雌激素和孕激素对体外培养奶牛破骨细胞增殖分化的影响[D]. 华中农业大学, 2007.Wu QR. Effects of estrogen and progesterone on the proliferation and differentiation of osteoclasts in dairy cows[D]. Huazhong Agricultural University, 2007.
[15] 付应霄, 顾建红, 赵鸿雁,等. 骨保护素对破骨细胞活性的影响[J]. 中国兽医学报, 2013, 33(5):738-741.Fu YX, Gu JH, Zhao HY, et al. Effect of osteoprotegerin on the activity of osteoclasts[J]. Chin J Vet Sci, 2013, 33(5):738-741.
[16] Kilic D, Kesim S, Liman N, et al. Comparison of the effects of dental filling Materials on mouse fibroblasts[J]. Biotechnol Biotechnol Eq, 2012, 26(4):3155-3162.
[17] Prause M, Seeliger C, Unger M, et al. Pantoprazole decreases cell viability and function of human osteoclasts in vitro[J]. Mediators Inflamm, 2015, 2015:413097.
[18] 李鹏辉, 田宗成, 商澎. 破骨细胞骨吸收功能的检测方法[J]. 生物学杂志, 2012, 29(4):74-77.Li PH, Tian ZC, Shang Y. Detection of bone resorption by osteoclasts[J]. J Biol, 2012, 29(4):74-77.