维生素D介导鸡BMSCs和BMMs共培养体系中破骨细胞的形成
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摘要
为探究1α, 25-(OH)_2D_3对鸡骨髓间充质干细胞(BMSCs)和骨髓单核巨噬细胞(BMMs)体外共培养体系中破骨细胞(OC)形成的影响,笔者对1α, 25-(OH)_2D_3处理后的细胞进行抗酒石酸酸性磷酸酶染色(TRAP)、骨吸收功能鉴定、OC标志基因mRNA和蛋白表达的检测。结果表明:1α, 25-(OH)_2D_3能够上调BMSCs中核因子κB受体活化因子配体(RANKL)的表达,下调骨保护素(OPG)的表达。1α, 25-(OH)_2D_3处理共培养细胞5 d,OC数目较对照组明显增多,骨吸收活性较对照组极显著增强,基质金属蛋白酶9(MMP-9)、Ⅱ型碳酸酐酶(CAⅡ)、组织蛋白酶K(CtsK)及TRAP等标志性蛋白的mRNA和蛋白表达极显著高于对照组(P<0.01),其中10~(-8) mol·L~(-1) 1α, 25-(OH)_2D_3效果最明显。结果表明,10~(-8) mol·L~(-1) 1α, 25-(OH)_2D_3能够介导鸡BMSCs和BMMs共培养体系中OC的形成,该OC具有骨吸收活性,为进一步研究OC在家禽骨骼及钙磷代谢紊乱性疾病中的作用机制奠定基础。
This study aimed to investigate the effect of 1α, 25-(OH)_2D_3 on the formation of osteoclast(OC) in the co-culture system of chicken bone marrow mesenchymal stem cells(BMSCs) and bone marrow mononuclear macrophage cells(BMMs). In the study, tartrate-resistant acid phosphatase(TRAP), bone absorption function identification, and OC marker mRNA and protein expression detection were performed on cells treated with 1α, 25-(OH)_2D_3. The results showed that, 1α, 25-(OH)_2D_3 up-regulated the expression of receptor activator for nuclear factor-κB ligand(RANKL) and down-regulated the expression of osteoprotegerin(OPG) of BMSCs. Compared to the control group, the number of OC was increased in the co-culture system at day 5 treated by 1α, 25-(OH)_2D_3. Meanwhile, the bone resorption activity, the mRNA and protein expression of matrix metalloproteinase 9(MMP-9), carbonic anhydrase Ⅱ(CAⅡ), cathepsin K and TRAP were significantly enhanced(P<0.01) by 1α, 25-(OH)_2D_3. Among them, 10~(-8) mol· L~(-1)1α, 25-(OH)_2D_3 had the most obvious effect. The results confirmed that 10~(-8) mol·L~(-1) 1α, 25-(OH)_2D_3 could mediate the formation of OC, which showed bone resorption activity, in the co-culture system of BMSCs and BMMs from chicken. Our study lays a foundation for further research on the mechanism of poultry OC in bone, calcium and phosphorus metabolic diseases.
This study aimed to investigate the effect of 1α, 25-(OH)_2D_3 on the formation of osteoclast(OC) in the co-culture system of chicken bone marrow mesenchymal stem cells(BMSCs) and bone marrow mononuclear macrophage cells(BMMs). In the study, tartrate-resistant acid phosphatase(TRAP), bone absorption function identification, and OC marker mRNA and protein expression detection were performed on cells treated with 1α, 25-(OH)_2D_3. The results showed that, 1α, 25-(OH)_2D_3 up-regulated the expression of receptor activator for nuclear factor-κB ligand(RANKL) and down-regulated the expression of osteoprotegerin(OPG) of BMSCs. Compared to the control group, the number of OC was increased in the co-culture system at day 5 treated by 1α, 25-(OH)_2D_3. Meanwhile, the bone resorption activity, the mRNA and protein expression of matrix metalloproteinase 9(MMP-9), carbonic anhydrase Ⅱ(CAⅡ), cathepsin K and TRAP were significantly enhanced(P<0.01) by 1α, 25-(OH)_2D_3. Among them, 10~(-8) mol· L~(-1)1α, 25-(OH)_2D_3 had the most obvious effect. The results confirmed that 10~(-8) mol·L~(-1) 1α, 25-(OH)_2D_3 could mediate the formation of OC, which showed bone resorption activity, in the co-culture system of BMSCs and BMMs from chicken. Our study lays a foundation for further research on the mechanism of poultry OC in bone, calcium and phosphorus metabolic diseases.
引文
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[22] GU J H,TONG X S,CHEN G H,et al.Regulation of matrix metalloproteinase-9 protein expression by 1α,25-(OH)2D3 during osteoclast differentiation[J].J Vet Sci,2014,15(1):133-140.
[23] CAPPARIELLO A,MAURIZI A,VEERIAH V,et al.The Great beauty of the osteoclast[J].Arch Biochem Biophys,2014,558:70-78.
[24] BOYCE B F,XING L P.The RANKL/RANK/OPG pathway[J].Curr Osteoporos Rep,2007,5(3):98-104.
[25] LIU W,ZHANG X L.Receptor activator of nuclear factor-κB ligand (RANKL)/RANK/osteoprotegerin system in bone and other tissues[J].Mol Med Rep,2015,11(5):3212-3218.
[26] CORRADO A,MARUOTTI N,CANTATORE F P.Osteoblast role in rheumatic diseases[J].Int J Mol Sci,2017,18(6):1272.
[27] PIRI F,KHOSRAVI A,MOAYERI A,et al.The effects of dietary supplements of calcium,vitamin D and estrogen hormone on serum levels of OPG and RANKL cytokines and their relationship with increased bone density in rats[J].J Clin Diagn Res,2016,10(9):AF01-AF04.
[28] WAN Q L,SCHOENMAKER T,JANSEN I D C,et al.Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone[J].Bone,2016,86:10-21.
[29] XU F,DONG Y H,HUANG X,et al.Pioglitazone affects the OPG/RANKL/RANK system and increase osteoclastogenesis[J].Mol Med Rep,2016,14(3):2289-2296.
[30] SONG C C,YANG X B,LEI Y S,et al.Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells[J].J Cell Physiol,2019,234(7):11969-11975.
[31] ZHU S,EHNERT S,ROUB M,et al.From the clinical problem to the basic research—co-culture models of osteoblasts and osteoclasts[J].Int J Mol Sci,2018,19(8):2284.
[32] 莫国业,张顺聪,李永贤,等.Transwell小室内建立小鼠成骨-破骨细胞共培养体系[J].中国骨伤,2018,31(3):241-247.MO G Y,ZHANG S C,LI Y X,et al.Establish mouse osteoblast-osteoclast cell co-culture system in a Transwell chamber[J].China Journal of Orthopaedics and Traumatology,2018,31(3):241-247.(in Chinese)
[33] KIM J H,KIM N.Signaling pathways in osteoclast differentiation[J].Chonnam Med J,2016,52(1):12-17.
[34] SAMBANDAM Y,SUNDARAM K,SAIGUSA T,et al.NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget’s disease of bone[J].Bone,2017,101:236-244.
[2] HOU Y C,WU C C,LIAO M T,et al.Role of nutritional vitamin D in osteoporosis treatment[J].Clin Chim Acta,2018,484:179-191.
[3] TAKAHASHI N,UDAGAWA N,SUDA T.Vitamin D endocrine system and osteoclasts[J].Bonekey Rep,2014,3:495.
[4] TSAI T L,LI W J.Identification of bone marrow-derived soluble factors regulating human mesenchymal stem cells for bone regeneration[J].Stem Cell Rep,2017,8(2):387-400.
[5] GENG S,ZHOU S H,BI Z G,et al.Vitamin D metabolism in human bone marrow stromal (mesenchymal stem) cells[J].Metabolism,2013,62(6):768-777.
[6] WANG D,GU J H,FENG L L,et al.1α,25-dihydroxyvitamin D3 potentiates avian osteoclast activation by increasing the formation of zipper-like structure via Src/Rac1 signaling[J].Biochem Biophys Res Commun,2018,501(2):576-583.
[7] CONG L,ZHANG C Y,TU G J.Osteoblastic NF-κB pathway is involved in 1α,25(OH)2D3-induced osteoclast-like cells formation in vitro[J].Int J Clin Exp Pathol,2015,8(5):5988-5996.
[8] GU J H,TONG X S,CHEN G H,et al.Effects of 1α,25-(OH)2D3 on the formation and activity of osteoclasts in RAW264.7 cells[J].J Steroid Biochem Mol Bio,2015,152:25-33.
[9] Kü?üKYILMAZ K,ERKEK R,BOZKURT M.The effects of boron supplementation of layer diets varying in calcium and phosphorus concentrations on performance,egg quality,bone strength and mineral constituents of serum,bone and faeces[J].Br Poult Sci,2014,55(6):804-816.
[10] 耿欢,累鸣,刘水涛,等.体外冲击波对骨髓源巨噬细胞向破骨细胞分化及骨吸收活性的影响[J].中国医学前沿杂志,2017,9(2):20-24.GENG H,LEI M,LIU S T,et al.The effect of extracorporeal shock wave on osteoclast formation of bone marrow-derived macrophage and bone resorption activity[J].Chinese Journal of the Frontiers of Medical Science (Electronic Version),2017,9(2):20-24.(in Chinese)
[11] DAI Q G,XIE F R,HAN Y J,et al.Inactivation of Regulatory-associated protein of mTOR (Raptor)/mammalian target of rapamycin complex 1 (mTORC1) signaling in osteoclasts increases bone mass by inhibiting osteoclast differentiation in mice[J].J Biol Chem,2017,292(1):196-204.
[12] ZHANG S,WANG X,LI G,et al.Osteoclast regulation of osteoblasts via RANK-RANKL reverse signal transduction in vitro[J].Mol Med Rep,2017,16(4):3994-4000.
[13] SIMON D,DERER A,ANDES F T,et al.Galectin-3 as a novel regulator of osteoblast-osteoclast interaction and bone homeostasis[J].Bone,2017,105:35-41.
[14] MCSHEEHY P M,CHAMBERS T J.1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption[J].J Clin Invest,1987,80(2):425-429.
[15] FIKADU W,TEGEGNE D,ABDELA N,et al.Milk fever and its economic consequences in dairy cows:a review[J].Global Vet,2016,16(5):441-452.
[16] CLARK W D,COX W R,SILVERSIDES F G.Bone fracture incidence in end-of-lay high-producing,noncommercial laying hens identified using radiographs[J].Poult Sci,2008,87(10):1964-1970.
[17] REGMI P,SMITH N,NELSON N,et al.Housing conditions alter properties of the tibia and humerus during the laying phase in Lohmann white Leghorn hens[J].Poult Sci,2016,95(1):198-206.
[18] 夏令.笼养蛋鸡产软壳蛋的原因及对策[J].现代畜牧科技,2018(8):34.XIA L.Causes and countermeasures of soft-shell eggs produced by caged layer hens[J].Modern Animal Husbandry Science & Technology,2018(8):34.(in Chinese)
[19] 王静,刘贵巧.蛋鸡产薄壳、软壳蛋的原因分析[J].中国畜牧兽医文摘,2017,33(4):96.WANG J,LIU G Q.Analysis of reasons for laying eggs with thin and soft shells[J].China Animal Husbandry & Veterinary Medicine Digest,2017,33(4):96.(in Chinese)
[20] TSUPRYKOV O,BUSE C,SKOBLO R,et al.Comparison of free and total 25-hydroxyvitamin D in normal human pregnancy[J].J Steroid Biochem Mol Biol,2019,190:29-36.
[21] MCVEY M K,GERAGHTY A A,O’BRIEN E C,et al.An exploratory analysis of associations of diet,sun exposure,and body composition with 25OHD at five years of age:findings from the ROLO Kids Study[J].J Steroid Biochem Mol Biol,2019,188:111-116.
[22] GU J H,TONG X S,CHEN G H,et al.Regulation of matrix metalloproteinase-9 protein expression by 1α,25-(OH)2D3 during osteoclast differentiation[J].J Vet Sci,2014,15(1):133-140.
[23] CAPPARIELLO A,MAURIZI A,VEERIAH V,et al.The Great beauty of the osteoclast[J].Arch Biochem Biophys,2014,558:70-78.
[24] BOYCE B F,XING L P.The RANKL/RANK/OPG pathway[J].Curr Osteoporos Rep,2007,5(3):98-104.
[25] LIU W,ZHANG X L.Receptor activator of nuclear factor-κB ligand (RANKL)/RANK/osteoprotegerin system in bone and other tissues[J].Mol Med Rep,2015,11(5):3212-3218.
[26] CORRADO A,MARUOTTI N,CANTATORE F P.Osteoblast role in rheumatic diseases[J].Int J Mol Sci,2017,18(6):1272.
[27] PIRI F,KHOSRAVI A,MOAYERI A,et al.The effects of dietary supplements of calcium,vitamin D and estrogen hormone on serum levels of OPG and RANKL cytokines and their relationship with increased bone density in rats[J].J Clin Diagn Res,2016,10(9):AF01-AF04.
[28] WAN Q L,SCHOENMAKER T,JANSEN I D C,et al.Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone[J].Bone,2016,86:10-21.
[29] XU F,DONG Y H,HUANG X,et al.Pioglitazone affects the OPG/RANKL/RANK system and increase osteoclastogenesis[J].Mol Med Rep,2016,14(3):2289-2296.
[30] SONG C C,YANG X B,LEI Y S,et al.Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells[J].J Cell Physiol,2019,234(7):11969-11975.
[31] ZHU S,EHNERT S,ROUB M,et al.From the clinical problem to the basic research—co-culture models of osteoblasts and osteoclasts[J].Int J Mol Sci,2018,19(8):2284.
[32] 莫国业,张顺聪,李永贤,等.Transwell小室内建立小鼠成骨-破骨细胞共培养体系[J].中国骨伤,2018,31(3):241-247.MO G Y,ZHANG S C,LI Y X,et al.Establish mouse osteoblast-osteoclast cell co-culture system in a Transwell chamber[J].China Journal of Orthopaedics and Traumatology,2018,31(3):241-247.(in Chinese)
[33] KIM J H,KIM N.Signaling pathways in osteoclast differentiation[J].Chonnam Med J,2016,52(1):12-17.
[34] SAMBANDAM Y,SUNDARAM K,SAIGUSA T,et al.NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget’s disease of bone[J].Bone,2017,101:236-244.