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Research Article

重组独特的软骨基质相关蛋白增强 MC3T3-E1 细胞的成骨分化和矿化

卷 22, 期 8, 2022

发表于: 14 January, 2022

页: [747 - 754] 页: 8

弟呕挨: 10.2174/1566524021666211117144314

价格: $65

摘要

目的：骨形成中成骨细胞和骨吸收中破骨细胞的相对平衡对于维持骨骼健康至关重要。随着年龄的增长，成骨细胞和破骨细胞之间的这种平衡被打破，导致骨质流失。正在不断开发合成代谢药物来抵消这种低骨量。重组蛋白被用作生物治疗药物，因为它相对容易大规模生产，并且通过各种表达系统具有成本效益。本研究旨在使用独特的软骨基质相关蛋白 (UCMA) 开发一种重组蛋白，该蛋白将对成骨细胞分化和矿化结节形成产生积极影响。 方法：在大肠杆菌系统中产生重组谷胱甘肽-S-转移酶（GST）-UCMA融合蛋白，并通过亲和层析纯化。将 MC3T3-E1 成骨细胞和 Osterix (Osx) 敲低稳定细胞培养 14 天，以研究在重组 GST-UCMA 蛋白存在下成骨细胞分化和结节形成。通过茜素红 S 染色和成骨细胞分化标志物骨钙素的定量 PCR 评估分化的细胞。此外，重组 GST-UCMA 蛋白存在下的细胞活力通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 测定和细胞粘附测定来确定。 结果：通过考马斯染色和蛋白质印迹分析，分别在 26 kDa 和 34 kDa 处证实了纯化的重组 GST-only 和 GST-UCMA 蛋白的分离。重组 GST-UCMA 的剂量依赖性和时间依赖性都不会影响 MC3T3-E1 细胞的活力。然而，MC3T3-E1 细胞对重组 GST-UCMA 蛋白的粘附呈剂量依赖性增加。在重组 GST-UCMA 蛋白存在下培养时，MC3T3-E1 成骨细胞和 Osxknockdown 稳定细胞均促进了成骨细胞分化和结节形成。 结论：重组 GST-UCMA 蛋白可诱导成骨分化和矿化，表明其可用作合成代谢药物以增加骨质疏松症患者的低骨量。

关键词: UCMA 蛋白、GST 融合、重组、成骨细胞、分化、矿化。

« Previous
[1]
Khan S, Ullah MW, Siddique R, et al. Role of recombinant DNA technology to improve life. Int J Genomics 2016; 2016: 2405954.
[http://dx.doi.org/10.1155/2016/2405954] [PMID: 28053975]
[2]
Reichert JM, Paquette C. Clinical development of therapeutic recombinant proteins. Biotechniques 2003; 35(1): 176-8.
[http://dx.doi.org/10.2144/03351dd01] [PMID: 12866419]
[3]
Wingfield PT. Overview of the purification of recombinant proteins. Curr Protoc Protein Sci 2015; 80: 6.1.1-6.1.35.
[http://dx.doi.org/10.1002/0471140864.ps0601s80]
[4]
Chen LR, Ko NY, Chen KH. Medical treatment for osteoporosis: From molecular to clinical opinions. Int J Mol Sci 2019; 20(9): 2213.
[http://dx.doi.org/10.3390/ijms20092213] [PMID: 31064048]
[5]
Ukon Y, Makino T, Kodama J, et al. Molecular-based treatment strategies for osteoporosis: A literature review. Int J Mol Sci 2019; 20(10): 2557.
[http://dx.doi.org/10.3390/ijms20102557] [PMID: 31137666]
[6]
Pavone V, Testa G, Giardina SMC, Vescio A, Restivo DA, Sessa G. Pharmacological therapy of osteoporosis: A systematic current review of literature. Front Pharmacol 2017; 8: 803.
[http://dx.doi.org/10.3389/fphar.2017.00803] [PMID: 29163183]
[7]
Seeman E, Martin TJ. Antiresorptive and anabolic agents in the prevention and reversal of bone fragility. Nat Rev Rheumatol 2019; 15(4): 225-36.
[http://dx.doi.org/10.1038/s41584-019-0172-3] [PMID: 30755735]
[8]
Liu Y, Levack AE, Marty E, et al. Anabolic agents: what is beyond osteoporosis? Osteoporos Int 2018; 29(5): 1009-22.
[http://dx.doi.org/10.1007/s00198-018-4507-8] [PMID: 29627891]
[9]
Russow G, Jahn D, Appelt J, Märdian S, Tsitsilonis S, Keller J. Anabolic therapies in osteoporosis and bone regeneration. Int J Mol Sci 2018; 20(1): 83.
[http://dx.doi.org/10.3390/ijms20010083] [PMID: 30587780]
[10]
Tagariello A, Luther J, Streiter M, et al. Ucma--A novel secreted factor represents a highly specific marker for distal chondrocytes. Matrix Biol 2008; 27(1): 3-11.
[http://dx.doi.org/10.1016/j.matbio.2007.07.004] [PMID: 17707622]
[11]
Surmann-Schmitt C, Dietz U, Kireva T, et al. Ucma, a novel secreted cartilage-specific protein with implications in osteogenesis. J Biol Chem 2008; 283(11): 7082-93.
[http://dx.doi.org/10.1074/jbc.M702792200] [PMID: 18156182]
[12]
Lee YJ, Park SY, Lee SJ, Boo YC, Choi JY, Kim JE. Ucma, a direct transcriptional target of Runx2 and Osterix, promotes osteoblast differentiation and nodule formation. Osteoarthritis Cartilage 2015; 23(8): 1421-31.
[http://dx.doi.org/10.1016/j.joca.2015.03.035] [PMID: 25865393]
[13]
Lee SJ, Lee EH, Park SY, Kim JE. Induction of fibrillin-2 and periostin expression in Osterix-knockdown MC3T3-E1 cells. Gene 2017; 596: 123-9.
[http://dx.doi.org/10.1016/j.gene.2016.10.018] [PMID: 27751812]
[14]
Kim SY, Lee EH, Park SY, et al. Ablation of stabilin-1 enhances bone-resorbing activity in osteoclasts in vitro. Calcif Tissue Int 2019; 105(2): 205-14.
[http://dx.doi.org/10.1007/s00223-019-00552-x] [PMID: 31025051]
[15]
Langdahl B, Ferrari S, Dempster DW. Bone modeling and remodeling: potential as therapeutic targets for the treatment of osteoporosis. Ther Adv Musculoskelet Dis 2016; 8(6): 225-35.
[http://dx.doi.org/10.1177/1759720X16670154] [PMID: 28255336]
[16]
Feng X, McDonald JM. Disorders of bone remodeling. Annu Rev Pathol 2011; 6: 121-45.
[http://dx.doi.org/10.1146/annurev-pathol-011110-130203] [PMID: 20936937]
[17]
Montagnani A. Bone anabolics in osteoporosis: Actuality and perspectives. World J Orthop 2014; 5(3): 247-54.
[http://dx.doi.org/10.5312/wjo.v5.i3.247] [PMID: 25035827]
[18]
Marie PJ. Transcription factors controlling osteoblasto-genesis. Arch Biochem Biophys 2008; 473(2): 98-105.
[http://dx.doi.org/10.1016/j.abb.2008.02.030] [PMID: 18331818]
[19]
Karsenty G, Kronenberg HM, Settembre C. Genetic control of bone formation. Annu Rev Cell Dev Biol 2009; 25: 629-48.
[http://dx.doi.org/10.1146/annurev.cellbio.042308.113308] [PMID: 19575648]
[20]
Raisz LG. Physiology and pathophysiology of bone remodeling. Clin Chem 1999; 45(8 Pt 2): 1353-8.
[PMID: 10430818]
[21]
Monroe DG, McGee-Lawrence ME, Oursler MJ, Westendorf JJ. Update on Wnt signaling in bone cell biology and bone disease. Gene 2012; 492(1): 1-18.
[http://dx.doi.org/10.1016/j.gene.2011.10.044] [PMID: 22079544]
[22]
Bodine PV, Komm BS. Wnt signaling and osteoblasto-genesis. Rev Endocr Metab Disord 2006; 7(1-2): 33-9.
[http://dx.doi.org/10.1007/s11154-006-9002-4] [PMID: 16960757]
[23]
Yang X, Yang Y, Zhou S, et al. Puerarin stimulates osteogenic differentiation and bone formation through the ERK1/2 and p38-MAPK signaling pathways. Curr Mol Med 2018; 17(7): 488-96.
[http://dx.doi.org/10.2174/1566524018666171219101142] [PMID: 29256352]
[24]
Kim WJ, Islam R, Kim BS, et al. Direct delivery of recombinant Pin1 protein rescued osteoblast differentiation of Pin1-deficient cells. J Cell Physiol 2017; 232(10): 2798-805.
[http://dx.doi.org/10.1002/jcp.25673] [PMID: 27800612]
[25]
Haas AV, LeBoff MS. Osteoanabolic agents for osteoporosis. J Endocr Soc 2018; 2(8): 922-32.
[http://dx.doi.org/10.1210/js.2018-00118] [PMID: 30087947]
[26]
Osagie-Clouard L, Sanghani A, Coathup M, Briggs T, Bostrom M, Blunn G. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017; 6(1): 14-21.
[http://dx.doi.org/10.1302/2046-3758.61.BJR-2016-0085.R1] [PMID: 28062525]
[27]
Yamashita J, McCauley LK. Effects of intermittent administration of parathyroid hormone and parathyroid hormone-related protein on fracture healing: A narrative review of animal and human studies. JBMR Plus 2019; 3(12): e10250.
[http://dx.doi.org/10.1002/jbm4.10250] [PMID: 31844831]
[28]
Neer RM, Arnaud CD, Zanchetta JR, et al. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 2001; 344(19): 1434-41.
[http://dx.doi.org/10.1056/NEJM200105103441904] [PMID: 11346808]
[29]
Miller PD, Hattersley G, Riis BJ, et al. Effect of abaloparatide vs placebo on new vertebral fractures in postmenopausal women with osteoporosis: A randomized clinical trial. JAMA 2016; 316(7): 722-33.
[http://dx.doi.org/10.1001/jama.2016.11136] [PMID: 27533157]
[30]
Leader B, Baca QJ, Golan DE. Protein therapeutics: A summary and pharmacological classification. Nat Rev Drug Discov 2008; 7(1): 21-39.
[http://dx.doi.org/10.1038/nrd2399] [PMID: 18097458]
[31]
Dimitrov DS. Therapeutic proteins. Methods Mol Biol 2012; 899: 1-26.
[http://dx.doi.org/10.1007/978-1-61779-921-1_1] [PMID: 22735943]
[32]
Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: Advances and challenges. Front Microbiol 2014; 5: 172.
[http://dx.doi.org/10.3389/fmicb.2014.00172] [PMID: 24860555]
[33]
Jia B, Jeon CO. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol 2016; 6(8): 160196.
[http://dx.doi.org/10.1098/rsob.160196] [PMID: 27581654]
[34]
Baeshen MN, Al-Hejin AM, Bora RS, et al. Production of biopharmaceuticals in E. coli: Current scenario and future perspectives. J Microbiol Biotechnol 2015; 25(7): 953-62.
[http://dx.doi.org/10.4014/jmb.1412.12079] [PMID: 25737124]
[35]
Landgraf W, Sandow J. Recombinant human insulins - Clinical efficacy and safety in Diabetes therapy. Eur Endocrinol 2016; 12(1): 12-7.
[http://dx.doi.org/10.17925/EE.2016.12.01.12] [PMID: 29632581]
[36]
Binkley N, Bone H, Gilligan JP, Krause DS. Efficacy and safety of oral recombinant calcitonin tablets in postmeno-pausal women with low bone mass and increased fracture risk: A randomized, placebo-controlled trial. Osteoporos Int 2014; 25(11): 2649-56.
[http://dx.doi.org/10.1007/s00198-014-2796-0] [PMID: 25027109]
[37]
Deng J, Feng Z, Li Y, Pan T, Li Q, Zhao C. Efficacy and safety of recombinant human parathyroid hormone (1-34) are similar to those of alendronate in the treatment of postmenopausal osteoporosis. Medicine (Baltimore) 2018; 97(47): e13341.
[http://dx.doi.org/10.1097/MD.0000000000013341] [PMID: 30461654]
[38]
Karsenty G, Ferron M. The contribution of bone to whole-organism physiology. Nature 2012; 481(7381): 314-20.
[http://dx.doi.org/10.1038/nature10763] [PMID: 22258610]
[39]
Patti A, Gennari L, Merlotti D, Dotta F, Nuti R. Endocrine actions of osteocalcin. Int J Endocrinol 2013; 2013: 846480.
[http://dx.doi.org/10.1155/2013/846480] [PMID: 23737779]
[40]
Zoch ML, Clemens TL, Riddle RC. New insights into the biology of osteocalcin. Bone 2016; 82: 42-9.
[http://dx.doi.org/10.1016/j.bone.2015.05.046] [PMID: 26055108]
[41]
Lin X, Parker L, Mclennan E, et al. Recombinant uncarboxylated osteocalcin per se enhances mouse skeletal muscle glucose uptake in both extensor digitorum longus and soleus muscles. Front Endocrinol (Lausanne) 2017; 8: 330.
[http://dx.doi.org/10.3389/fendo.2017.00330] [PMID: 29204135]
[42]
Käkönen SM, Hellman J, Pettersson K, Lövgren T, Karp M. Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli. Protein Expr Purif 1996; 8(2): 137-44.
[http://dx.doi.org/10.1006/prep.1996.0085] [PMID: 8812845]
[43]
Kim JH, Park S, Kim HW, Jang JH. Recombinant expression of mouse osteocalcin protein in Escherichia coli. Biotechnol Lett 2007; 29(11): 1631-5.
[http://dx.doi.org/10.1007/s10529-007-9437-z] [PMID: 17598072]
[44]
Le Jeune M, Tomavo N, Tian TV, et al. Identification of four alternatively spliced transcripts of the Ucma/GRP gene, encoding a new Gla-containing protein. Exp Cell Res 2010; 316(2): 203-15.
[http://dx.doi.org/10.1016/j.yexcr.2009.10.002] [PMID: 19819238]
[45]
Tesfamariam B. Involvement of vitamin K-dependent proteins in vascular calcification. J Cardiovasc Pharmacol Ther 2019; 24(4): 323-33.
[http://dx.doi.org/10.1177/1074248419838501] [PMID: 30905168]
[46]
Wen L, Chen J, Duan L, Li S. Vitamin K dependent proteins involved in bone and cardiovascular health (Review). Mol Med Rep 2018; 18(1): 3-15.
[http://dx.doi.org/10.3892/mmr.2018.8940] [PMID: 29749440]
[47]
Willems BA, Furmanik M, Caron MMJ, et al. Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling. Sci Rep 2018; 8(1): 4961.
[http://dx.doi.org/10.1038/s41598-018-23353-y] [PMID: 29563538]
[48]
Stock M, Menges S, Eitzinger N, et al. A dual role of upper zone of growth plate and cartilage matrix-associated protein in human and mouse osteoarthritic cartilage: Inhibition of aggrecanases and promotion of bone turnover. Arthritis Rheumatol 2017; 69(6): 1233-45.
[http://dx.doi.org/10.1002/art.40042] [PMID: 28086000]
[49]
Lee SH, Lee YJ, Park SI, Kim JE. Unique cartilage matrix-associated protein inhibits the migratory and invasive potential of triple-negative breast cancer. Biochem Biophys Res Commun 2020; 530(4): 680-5.
[http://dx.doi.org/10.1016/j.bbrc.2020.07.114] [PMID: 32768190]
[50]
Nakashima K, Zhou X, Kunkel G, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002; 108(1): 17-29.
[http://dx.doi.org/10.1016/S0092-8674(01)00622-5] [PMID: 11792318]

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