Effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHENG Libin ¹ ,  HUANG Dong ^1,2

1. Guangdong Medical University, Zhanjiang Guangdong, 524000, P.R.China;
2. Department of Trauma Orthopedics, Guangdong Second Provincial Central Hospital, Guangzhou Guangdong, 510000, P.R.China;

Corresponding author：

HUANG Dong, Email: dong-177@163.com

Keywords：

FTY720-P; MC3T3-E1 cells; osteoblasts; cell apoptosis

DOI：

10.7507/1002-1892.201710108

Video：

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ObjectiveTo investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells.MethodsThe MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.ResultsAfter 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance (IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) (t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups (t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) (t=23.420, P=0.002).ConclusionFTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.

Citation： ZHENG Libin, HUANG Dong. Effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(3): 285-290. doi: 10.7507/1002-1892.201710108 Copy

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2.	Ettenger R, Schmouder R, Kovarik JM, et al. Pharmacokinetics, pharmacodynamics, safety, and tolerability of single-dose fingolimod (FTY720) in adolescents with stable renal transplants. Pediatr Transplant, 2015, 15(4): 406-413.
3.	熊苗, 朱洁萍, 李莉, 等. 过继转移 FTY720-DC 对自然流产模型孕鼠胚胎丢失率的影响. 上海交通大学学报(医学版), 2016, 36(6): 793-798.
4.	王燕, 扈金萍. 鞘氨醇-1-磷酸转运体 Spns2 的研究进展. 国际药学研究杂志, 2016, 43(6): 1043-1048.
5.	白洁, 扈金萍. 鞘氨醇-1-磷酸信号通路相关的药物研究进展. 药学学报, 2016, 51(12): 1822-1828.
6.	Zhang D, Huang Y, Huang Z, et al. FTY-720P suppresses osteoclast formation by regulating expression of interleukin-6(IL-6), interleukin-4(IL-4), and matrix metalloproteinase 2(MMP-2). Med Sci Monit, 2016, 22: 2187-2194.
7.	陈智颖, 左洁仪, 兰万利, 等. FTY720-P 对成骨细胞分化成熟的作用. 广东医学, 2017, 38(13): 1957-1961.
8.	Leis HJ, Windischhofer W. Potentiation of endothelin-1-induced prostaglandin E2 formation by Ni (2+) and Sr (2+) in murine osteoblastic MC3T3-E1 cells. J Trace Elem Med Biol, 2016, 33: 81-86.
9.	Bueno-Vera JA, Torres-Zapata I, Sundaram PA, et al. Electrochemical characterization of MC3T3-E1 cells cultured on γTiAl and Ti-6Al-4V alloys. Bioelectrochemistry, 2015, 106(Pt B): 316-327.
10.	Shinomiya T, Li XK, Amemiya H, et al. An immunosuppressive agent, FTY720, increases in tracellular concentration of calcium ion and induces apoptosis in HL-60. Immunology, 1997, 91(4): 594-600.
11.	Wang Z, Hu H, Li Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofacial Surg, 2016, 54(3): 316-321.
12.	Xu X, Gao Y, Shan F, et al. A novel role for RGMa in modulation of bone marrow-derived dendritic cells maturation induced by lipopolysaccharide. Int Immunopharmacol, 2016, 33: 99-107.
13.	Guo J, Watterson SH, Spergel SH, et al. Identification and synthesis of potent and selective pyridyl-isoxazole based agonists of sphingosine-1-phosphate 1(S1P1). Bioorg Med Chem Lett, 2016, 26(10): 2470-2474.
14.	郭海玲, 王翔, 徐宇, 等. 黄芪调控体外培养大鼠成骨细胞 Ⅰ 型胶原蛋白的表达. 中国组织工程研究, 2010, 14(7): 1257-1261.
15.	Ho MH, Liao MH, Lin YL, et al. Improving effects of chitosannanofiber scaffolds on osteoblast proliferation and maturation. Int J Nanomedicine, 2014, 9: 4293-4304.
16.	Izu Y, Ezura Y, Koch M, et al. Erratum to: Collagens Ⅵ and Ⅻ form complexes mediating osteoblast interactions during osteogenesis. Cell Tissue Res, 2016, 364(3): 677-679.
17.	Das A, Barker DA, Wang T, et al. Delivery of bioactive lipids from composite microgel-microsphere injectable scaffolds enhances stem cell recruitment and skeletal repair. PLoS One, 2014, 9(7): e101276.
18.	Liu Zhonghou. The diagnosis of osteoporosis. Hong Kong: Chinese modern literature and Art Publishing House, 2016: 452-465.
19.	Matsugaki A, Fujiwara N, Nakano T. Continuous cyclic stretch induces osteoblast alignment and formation of anisotropic collagen fiber matrix. Acta Biomater, 2013, 3(19): 1878-1889.
20.	Dehaghani MT, Ahmadian M. Porous vitalium-base nano-composite for bone replacement: Fabrication, mechanical, and in vitro biological properties. J Mech Behav Biomed Mater, 2016, 57: 297-309.
21.	朱文辉, 陈世益. 肌球蛋白重链及 Ⅰ、Ⅲ 型胶原在骨骼肌损伤修复中的作用. 中国运动医学杂志, 2002, 21(2): 179-182.

1. Brinkmann V, Davis MD, Heise CE, et al. The immunemodulator FTY720 targets sphingosine 1-phosphatereceptors. J Biol Chem, 2002, 277(24): 21453-21457.
2. Ettenger R, Schmouder R, Kovarik JM, et al. Pharmacokinetics, pharmacodynamics, safety, and tolerability of single-dose fingolimod (FTY720) in adolescents with stable renal transplants. Pediatr Transplant, 2015, 15(4): 406-413.
3. 熊苗, 朱洁萍, 李莉, 等. 过继转移 FTY720-DC 对自然流产模型孕鼠胚胎丢失率的影响. 上海交通大学学报(医学版), 2016, 36(6): 793-798.
4. 王燕, 扈金萍. 鞘氨醇-1-磷酸转运体 Spns2 的研究进展. 国际药学研究杂志, 2016, 43(6): 1043-1048.
5. 白洁, 扈金萍. 鞘氨醇-1-磷酸信号通路相关的药物研究进展. 药学学报, 2016, 51(12): 1822-1828.
6. Zhang D, Huang Y, Huang Z, et al. FTY-720P suppresses osteoclast formation by regulating expression of interleukin-6(IL-6), interleukin-4(IL-4), and matrix metalloproteinase 2(MMP-2). Med Sci Monit, 2016, 22: 2187-2194.
7. 陈智颖, 左洁仪, 兰万利, 等. FTY720-P 对成骨细胞分化成熟的作用. 广东医学, 2017, 38(13): 1957-1961.
8. Leis HJ, Windischhofer W. Potentiation of endothelin-1-induced prostaglandin E2 formation by Ni (2+) and Sr (2+) in murine osteoblastic MC3T3-E1 cells. J Trace Elem Med Biol, 2016, 33: 81-86.
9. Bueno-Vera JA, Torres-Zapata I, Sundaram PA, et al. Electrochemical characterization of MC3T3-E1 cells cultured on γTiAl and Ti-6Al-4V alloys. Bioelectrochemistry, 2015, 106(Pt B): 316-327.
10. Shinomiya T, Li XK, Amemiya H, et al. An immunosuppressive agent, FTY720, increases in tracellular concentration of calcium ion and induces apoptosis in HL-60. Immunology, 1997, 91(4): 594-600.
11. Wang Z, Hu H, Li Z, et al. Sheet of osteoblastic cells combined with platelet-rich fibrin improves the formation of bone in critical-size calvarial defects in rabbits. Br J Oral Maxillofacial Surg, 2016, 54(3): 316-321.
12. Xu X, Gao Y, Shan F, et al. A novel role for RGMa in modulation of bone marrow-derived dendritic cells maturation induced by lipopolysaccharide. Int Immunopharmacol, 2016, 33: 99-107.
13. Guo J, Watterson SH, Spergel SH, et al. Identification and synthesis of potent and selective pyridyl-isoxazole based agonists of sphingosine-1-phosphate 1(S1P1). Bioorg Med Chem Lett, 2016, 26(10): 2470-2474.
14. 郭海玲, 王翔, 徐宇, 等. 黄芪调控体外培养大鼠成骨细胞 Ⅰ 型胶原蛋白的表达. 中国组织工程研究, 2010, 14(7): 1257-1261.
15. Ho MH, Liao MH, Lin YL, et al. Improving effects of chitosannanofiber scaffolds on osteoblast proliferation and maturation. Int J Nanomedicine, 2014, 9: 4293-4304.
16. Izu Y, Ezura Y, Koch M, et al. Erratum to: Collagens Ⅵ and Ⅻ form complexes mediating osteoblast interactions during osteogenesis. Cell Tissue Res, 2016, 364(3): 677-679.
17. Das A, Barker DA, Wang T, et al. Delivery of bioactive lipids from composite microgel-microsphere injectable scaffolds enhances stem cell recruitment and skeletal repair. PLoS One, 2014, 9(7): e101276.
18. Liu Zhonghou. The diagnosis of osteoporosis. Hong Kong: Chinese modern literature and Art Publishing House, 2016: 452-465.
19. Matsugaki A, Fujiwara N, Nakano T. Continuous cyclic stretch induces osteoblast alignment and formation of anisotropic collagen fiber matrix. Acta Biomater, 2013, 3(19): 1878-1889.
20. Dehaghani MT, Ahmadian M. Porous vitalium-base nano-composite for bone replacement: Fabrication, mechanical, and in vitro biological properties. J Mech Behav Biomed Mater, 2016, 57: 297-309.
21. 朱文辉, 陈世益. 肌球蛋白重链及 Ⅰ、Ⅲ 型胶原在骨骼肌损伤修复中的作用. 中国运动医学杂志, 2002, 21(2): 179-182.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells

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