人源瓣膜间质细胞体外成骨诱导方法比较

doi:10.6040/j.issn.1671-7554.0.2024.0701

摘要/Abstract

摘要： 目的探究不同成骨诱导方法对人源瓣膜间质细胞(human valvular interstitial cells, hVICs)成骨分化及钙化的影响。方法选取常用的5种体外成骨诱导瓣膜间质细胞方法对hVICs进行成骨诱导分化:(1) 2 mmol/L无机磷酸盐+50 μg/mL维生素C(2 mmol/L Pi+VC);(2) 2.6 mmol/L无机磷酸盐(2.6 mmol/L Pi);(3) 2.5 mmol/L无机磷酸盐+2.7 mmol/L氯化钙+50 μg/mL维生素C(2.5 mmol/L Pi+Ca+VC);(4) 10 mmol/L β-磷酸甘油+10 nmol/L地塞米松+8 mmol/L氯化钙+4 μg/mL维生素D3(β-GP+DXM+Ca+VD3);(5) 10 mmol/L β-磷酸甘油+10 nmol/L地塞米松+50 μg/mL维生素C(β-GP+DXM+VC)。通过茜素红染色、RT-qPCR和Western blotting分别对钙化沉积和成骨标志物的表达进行评估。结果通过比较5种诱导方法在成骨诱导第4、7、10、14 d的钙沉积面积,发现2 mmol/L Pi+VC、β-GP+DXM+Ca+VD3和β-GP+DXM+VC在前10 d几乎没有钙化,而2.6 mmol/L Pi在第7天出现明显钙化,2.5 mmol/L Pi+Ca+VC在诱导第4天便出现明显钙化,且在第7天时几乎全部发生钙化。在第14天时,除2 mmol/L Pi+VC 钙化较少外,其余方法均发生明显钙化。成骨标志物检测显示,碱性磷酸酶在2 mmol/L Pi+VC、2.6 mmol/L Pi和β-GP+DXM+VC诱导第7天时显著上调(P<0.05);而骨桥蛋白在诱导第7天时均显著上调(P<0.001)。随后对2.5 mmol/L Pi+Ca+VC中的3种组分两两组合进行钙化诱导,发现Pi+Ca和Pi+Ca+VC在诱导第7天时钙化面积显著多于Pi+VC和Ca+VC,但ALP表达显著低于Pi+VC和Ca+VC,提示这种钙沉积可能是由于无机磷酸盐与氯化钙发生反应生成了磷酸钙沉淀。结论 2.6 mmol/L Pi和β-GP+DXM+VC诱导hVICs成骨分化的效果最佳。

关键词: 瓣膜间质细胞, 成骨诱导, 成骨分化, 钙化, 无机磷酸盐, β-磷酸甘油

Abstract: Objective To explore the effects of different osteogenic induction methods on the osteogenic differentiation and calcification of human valvular interstitial cells(hVICs). Methods Five commonly used in vitro osteogenic induction methods for valvular interstitial cells were selected from the literature: (1) 2 mmol/L inorganic phosphate+50 μg/mL vitamin C(2 mmol/L Pi + VC); (2) 2.6 mmol/L inorganic phosphate(2.6 mmol/L Pi); (3) 2.5 mmol/L inorganic phosphate+2.7 mmol/L calcium chloride+50 μg/mL vitamin C(2.5 mmol/L Pi + Ca + VC); (4) 10 mmol/L β-glycerophosphate+10 nmol/L dexamethasone+8 mmol/L calcium chloride+4 μg/mL vitamin D3(β-GP+DXM+Ca+VD3); (5) 10 mmol/L β-glycerophosphate+10 nmol/L dexamethasone+50 μg/mL vitamin C(β-GP+DXM+VC). Alizarin red staining, RT-qPCR and Western blotting were used to assess calcium deposition and the expression of osteogenic markers, respectively. Results By comparing the calcium deposition areas on day 4, 7, 10, and 14 induced by five different induction methods, it was found that there was almost no calcification within the first 10 days with 2 mmol/L Pi+VC, β-GP+DXM+Ca+VD3, and β-GP+DXM+VC. In contrast, significant calcification appeared on day 7 with 2.6 mmol/L Pi, 2.5 mmol/L Pi+Ca+VC showed significant calcification on day 4 and almost all calcification on day 7. On day 14, except for the relatively low calcification with 2 mmol/L Pi+VC, all other methods showed significant calcification. Osteogenic marker detection indicated that alkaline phosphatase was significantly upregulated on day 7 with 2 mmol/L Pi+VC, 2.6 mmol/L Pi, and β-GP+DXM+VC(P<0.05), while osteopontin showed significant upregulation on day 7 in all methods(P<0.001). Subsequently, calcium induction was performed with pairwise combinations of the three components in 2.5 mmol/L Pi+Ca+VC. It was found that the calcium deposition areas were significantly larger with Pi+Ca and Pi+Ca+VC on day 7 compared to Pi+VC and Ca+VC, but ALP expression was significantly lower in Pi+Ca and Pi+Ca+VC, suggesting that this calcium deposition might be due to the reaction between inorganic phosphate and calcium chloride forming calcium phosphate precipitates. Conclusion The optimal effect of inducing osteogenic differentiation in hVICs was achieved with 2.6 mmol/L Pi and β-GP+DXM+VC.

Key words: Valvular interstitial cell, Osteogenic induction, Osteogenic differentiation, Calcification, Inorganic phosphate, β-glycerophosphate

中图分类号:

R331

邢凯,郑强,孙金书,刘晓林,王正军. 人源瓣膜间质细胞体外成骨诱导方法比较[J]. 山东大学学报 (医学版), 2025, 63(6): 11-18.

XING Kai, ZHENG Qiang, SUN Jinshu, LIU Xiaolin, WANG Zhengjun. Comparison of osteogenic induction methods for human valve interstitial cells in vitro[J]. Journal of Shandong University (Health Sciences), 2025, 63(6): 11-18.

参考文献

[1] Bogdanova M, Zabirnyk A, Malashicheva A, et al. Models and techniques to study aortic valve calcification in vitro, ex vivo and in vivo. an overview[J]. Front Pharmacol, 2022, 13: 835825. doi:10.3389/fphar.2022.835825
[2] Coffey S, Roberts-Thomson R, Brown A, et al. Global epidemiology of valvular heart disease[J]. Nat Rev Cardiol, 2021, 18(12): 853-864.
[3] Pinto G, Fragasso G. Aortic valve stenosis: drivers of di-sease progression and drug targets for therapeutic opportunities[J]. Expert Opin Ther Targets, 2022, 26(7): 633-644.
[4] de Oliveira Sá MPB, Cavalcanti LRP, Perazzo áM, et al. Calcific aortic valve stenosis and atherosclerotic calcification[J]. Curr Atheroscler Rep, 2020, 22(2): 2. doi:10.1007/s11883-020-0821-7
[5] Goody PR, Hosen MR, Christmann D, et al. Aortic valve stenosis[J]. Arterioscler Thromb Vasc Biol, 2020, 40(4): 885-900.
[6] Moncla LM, Briend M, Bossé Y, et al. Calcific aortic valve disease: mechanisms, prevention and treatment[J]. Nat Rev Cardiol, 2023, 20(8): 546-559.
[7] 薛清, 李宁, 褚恒, 等. 有机磷酸盐-与无机磷酸盐-钙化诱导培养基诱导主动脉瓣膜间质细胞钙化效果的对比研究[J]. 实用心脑肺血管病杂志, 2018, 26(4): 44-49. XUE Qing, LI Ning, CHU Heng, et al. Comparative study for effect on aortic valve interstitial cells calcification between organic phosphate- and inorganic phosphate-osteogenic induction medium[J]. Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease, 2018, 26(4): 44-49.
[8] Niepmann ST, Willemsen N, Boucher AS, et al. Toll-like receptor-3 contributes to the development of aortic valve stenosis[J]. Basic Res Cardiol, 2023, 118(1): 6. doi:10.1007/s00395-023-00980-9
[9] Zheng KH, Tsimikas S, Pawade T, et al. Lipoprotein(a)and oxidized phospholipids promote valve calcification in patients with aortic stenosis[J]. J Am Coll Cardiol, 2019, 73(17): 2150-2162.
[10] Qiao E, Huang ZP, Wang YT, et al. Metformin alle-viates the calcification of aortic valve interstitial cells through activating the PI3K/AKT pathway in an AMPK dependent way[J]. Mol Med, 2021, 27(1): 156. doi:10.1186/s10020-021-00416-x
[11] Éva Sikura K, Combi Z, Potor L, et al. Hydrogen sulfide inhibits aortic valve calcification in heart via regulating RUNX2 by NF-κB, a link between inflammation and mineralization[J]. J Adv Res, 2020, 27: 165-176. doi:10.1016/j.jare.2020.07.005
[12] Jover E, Matilla L, Martín-Núñez E, et al. Sex-dependent expression of neutrophil gelatinase-associated lipocalin in aortic stenosis[J]. Biol Sex Differ, 2022, 13(1): 71. doi:10.1186/s13293-022-00480-w
[13] Artiach G, Carracedo M, Plunde O, et al. Omega-3 polyunsaturated fatty acids decrease aortic valve disease through the resolvin E1 and ChemR23 axis[J]. Circulation, 2020, 142(8): 776-789.
[14] Chen Z, Gordillo-Martinez F, Jiang L, et al. Zinc ame-liorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway[J]. Cardiovasc Res, 2021, 117(3): 820-835.
[15] Liu Z, Dong N, Hui H, et al. Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification[J]. Eur Heart J, 2022, 43(17): 1652-1664.
[16] Zhong GH, Su SW, Li JC, et al. Activation of Piezo1 promotes osteogenic differentiation of aortic valve interstitial cell through YAP-dependent glutaminolysis[J]. Sci Adv, 2023, 9(22): eadg0478. doi:10.1126/sciadv.adg0478
[17] Li SY, Luo ZC, Su SW, et al. Targeted inhibition of PTPN22 is a novel approach to alleviate osteogenic responses in aortic valve interstitial cells and aortic valve lesions in mice[J]. BMC Med, 2023, 21(1): 252. doi:10.1186/s12916-023-02888-6
[18] Iqbal F, Schlotter F, Becker-Greene D, et al. Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity[J]. Eur Heart J, 2023, 44(10): 885-898.
[19] Grim JC, Aguado BA, Vogt BJ, et al. Secreted factors from proinflammatory macrophages promote an osteoblast-like phenotype in valvular interstitial cells[J]. Arterioscler Thromb Vasc Biol, 2020, 40(11): e296-e308.
[20] Sánchez-Esteban S, Castro-Pinto M, Cook-Calvete A, et al. Integrin-linked kinase expression in human valve endothelial cells plays a protective role in calcific aortic valve disease[J]. Antioxidants, 2022, 11(9): 1736. doi:10.3390/antiox11091736
[21] Liu F, Chen J, Hu W, et al. PTP1B inhibition improves mitochondrial dynamics to alleviate calcific aortic valve disease via regulating OPA1 homeostasis[J]. JACC Basic Transl Sci, 2022, 7(7): 697-712.
[22] Shu L, Yuan Z, Li F, et al. Oxidative stress and valvular endothelial cells in aortic valve calcification[J]. Biomed Pharmacother, 2023, 163: 114775. doi:10.1016/j.biopha.2023.114775
[23] Sud K, Narula N, Aikawa E, et al. The contribution of amyloid deposition in the aortic valve to calcification and aortic stenosis[J]. Nat Rev Cardiol, 2023, 20(6): 418-428.
[24] Summerhill VI, Moschetta D, Orekhov AN, et al. Sex-specific features of calcific aortic valve disease[J]. Int J Mol Sci, 2020, 21(16): E5620. doi:10.3390/ijms21165620
[25] Ternacle J, Krapf L, Mohty D, et al. Aortic stenosis and cardiac amyloidosis[J]. J Am Coll Cardiol, 2019, 74(21): 2638-2651.
[26] Villa-Bellosta R. Vascular calcification: key roles of phosphate and pyrophosphate[J]. Int J Mol Sci, 2021, 22(24): 13536. doi:10.3390/ijms222413536
[27] Alushi B, Curini L, Christopher MR, et al. Calcific aortic valve disease-natural history and future therapeutic strategies[J]. Front Pharmacol, 2020, 11: 685. doi:10.3389/fphar.2020.00685
[28] Leopold JA. Cellular mechanisms of aortic valve calcification[J]. Circulation: Cardiovascular Interventions, 2012, 5(4): 605-614.
[29] Wehner C, Lettner S, Moritz A, et al. Effect of bisphosphonate treatment of titanium surfaces on alkaline phosphatase activity in osteoblasts: a systematic review and meta-analysis[J]. BMC Oral Health, 2020, 20(1): 125. doi: 10.1186/s12903-020-01089-4
[30] Chatakun P, Núñez-Toldrà R, Díaz López EJ, et al. The effect of five proteins on stem cells used for osteoblast differentiation and proliferation: a current review of the literature[J]. Cell Mol Life Sci, 2014, 71(1): 113-142.

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