Effects of daphnetin combined with insulin-like growth factor 1 on chondrogenic differentiation of adipose-derived mesenchymal stem cells in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SHEN Ning ¹ , XI Xiaoyun ¹ , LI Fei ² , LÜ Chaoliang ³ ,  ZHANG Pusheng ¹

1. Department of Orthopedics, Jinxiang Hospital of Jining Medical University, Jining Shandong, 272200, P.R.China;
2. Department of Radiology, Jinxiang Hospital of Jining Medical University, Jining Shandong, 272200, P.R.China;
3. Department of Orthopedics, Jining First People’s Hospital, Jining Shandong, 272200, P.R.China;

Corresponding author：

ZHANG Pusheng, Email: jinxiangsn@126.com

Keywords：

Adipose-derived mesenchymal stem cells; daphnetin; insulin-like growth factor 1; chondrogenic differentiation; rat

DOI：

10.7507/1002-1892.201812063

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Objective To investigate the effect of daphnetin (DAP) combined with insulin-like growth factor 1 (IGF-1) gene transfection on chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) in rats.Methods Rat ADSCs were isolated and amplified by enzymatic digestion. The third generation ADSCs were treated with IGF-1 gene transfection as experimental group and normal ADSCs as control group. The cells of the two groups were treated with different concentrations of DAP (0, 30, 60, 90 μg/mL), respectively. Cell proliferation was detected by cell counting kit 8 (CCK-8) after cultured for 72 hours. After 14 days, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of chondrocyte markers (collagen type Ⅱ and Aggrecan) in each group; and toluidine blue staining and collagen type Ⅱ immunohistochemical staining were performed.Results CCK-8 assay showed that with the increase of DAP concentration, the cell absorbance (A) value of the control group and the experimental group increased gradually (P<0.05). At the same DAP concentration, the cell A value of the experimental group was significantly higher than that of the control group (P<0.05). Real-time fluorescence quantitative PCR and Western blot showed that with the increase of DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the control group did not change significantly, and there was no significant difference among the different concentration groups (P>0.05). But the mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the experimental group increased gradually, and the 60 and 90 μg/mL DAP concentration groups were significantly higher than 0 μg/mL DAP concentration group (P<0.05). At the same DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan were significantly higher in the experimental group than in the control group (P<0.05). Toluidine blue staining showed that with the increase of DAP concentration, there was no significant difference in cell staining between the control group and the experimental group. At the same DAP concentration, the cells in the experimental group were slightly darker than those in the control group. Immunohistochemical staining of collagen type Ⅱ showed that with the increase of DAP concentration, there was no significant difference in the cytoplasmic brown-yellow coloring of the cells in the control group. The cytoplasmic brown-yellow coloring of the cells in the experimental group gradually deepened, with 60 and 90 μg/mL DAP concentration groups significantly deeper than 0 μg/mL DAP concentration group. At the same DAP concentration, the color of the cells in the experimental group was significantly deeper than that in the control group.Conclusion DAP can promote the proliferation of ADSCs in rats. The differentiation of ADSCs into chondrocytes induced by DAP alone was slightly, but DAP combined with IGF-1 gene transfection has obvious synergistic effect to promote chondrogenic differentiation of ADSCs.

Citation： SHEN Ning, XI Xiaoyun, LI Fei, LÜ Chaoliang, ZHANG Pusheng. Effects of daphnetin combined with insulin-like growth factor 1 on chondrogenic differentiation of adipose-derived mesenchymal stem cells in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(6): 743-749. doi: 10.7507/1002-1892.201812063 Copy

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3.	宋克东, 杨延飞, 李文芳, 等. ADSCs-壳聚糖/明胶水凝胶工程化软骨的三维动态构建. 功能材料, 2015, 46(15): 15021-15030.
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7.	Tu L, Li S, Fu Y, et al. The therapeutic effects of daphnetin in collagen-induced arthritis involve its regulation of Th17 cells. Int Immunopharmacol, 2012, 13(4): 417-423.
8.	朱国华, 齐新生. 关节软骨缺损修复的实验与临床. 中国骨伤, 2004, 17(5): 318-320.
9.	刘振宁, 贾长青, 韩长旭, 等. 生长分化因子 5 诱导兔脂肪干细胞成软骨细胞分化的实验研究. 中国修复重建外科杂志, 2009, 23(4): 483-489.
10.	Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 2001, 7(2): 211-228.
11.	史琳丽, 杨向群. 脂肪组织来源干细胞的分化潜能和应用. 中国修复重建外科杂志, 2012, 26(8): 1007-1011.
12.	Erickson GR, Gimble JM, Franklin DM, et al. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun, 2002, 290(2): 763-769.
13.	Messai H, Duchossoy Y, Khatib AM, et al. Articular chondrocytes from aging rats respond poorly to insulin-like growth factor-1: an altered signaling pathway. Mech Ageing Dev, 2000, 115(1-2): 21-37.
14.	杨强, 丁晓明, 徐宝山, 等. 取向性丝素蛋白支架复合脂肪干细胞体外构建组织工程软骨. 中华骨科杂志, 2015, 35(12): 1235-1242.
15.	An C, Cheng Y, Yuan Q, et al. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng, 2010, 38(4): 1647-1654.
16.	Zheng W, Gao X, Gu Q, et al. Antitumor activity of daphnodorins from Daphne genkwa roots. Int Immunopharmacol, 2007, 7(2): 128-134.
17.	Prince M, Li Y, Childers A, et al. Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice. Toxicol Lett, 2009, 185(3): 180-186.
18.	Cottiglia F, Loy G, Garau D, et al. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium L. Phytomedicine, 2001, 8(4): 302-305.
19.	邹文玮, 舒奇, 傅颖媛, 等. 瑞香素毒理学实验研究. 中成药, 2013, 35(5): 908-911.
20.	Gao Q, Shan J, Di L, et al. Therapeutic effects of daphnetin on adjuvant-induced arthritic rats. J Ethnopharmacol, 2008, 120(2): 259-263.
21.	Tang DZ, Hou W, Zhou Q, et al. Osthole stimulates osteoblast differentiation and bone formation by activation of beta-catenin-BMP signaling. J Bone Miner Res, 2010, 25(6): 1234-1245.
22.	Liu S, Shao Y, Lin Q, et al. 7, 8-Dihydroxy coumarin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells. J Int Med Res, 2013, 41(1): 82-96.

1. Redman SN, Oldfield SF, Archer CW. Current strategies for articular cartilage repair. Eur Cell Mater, 2005, 9: 23-32.
2. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002, 13(12): 4279-4295.
3. 宋克东, 杨延飞, 李文芳, 等. ADSCs-壳聚糖/明胶水凝胶工程化软骨的三维动态构建. 功能材料, 2015, 46(15): 15021-15030.
4. 张勇, 陈晓菊, 王燕杰, 等. 兔脂肪干细胞直接修复陈旧性全层兔膝关节软骨缺损. 临床骨科杂志, 2015, 18(4): 492-497.
5. Longobardi L, O’Rear L, Aakula S, et al. Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling. J Bone Miner Res, 2006, 21(4): 626-636.
6. 赵明璨, 刘畅. 脂肪干细胞在软骨组织工程中的研究进展. 中国生物医学工程学报, 2009, 33(4): 475-481.
7. Tu L, Li S, Fu Y, et al. The therapeutic effects of daphnetin in collagen-induced arthritis involve its regulation of Th17 cells. Int Immunopharmacol, 2012, 13(4): 417-423.
8. 朱国华, 齐新生. 关节软骨缺损修复的实验与临床. 中国骨伤, 2004, 17(5): 318-320.
9. 刘振宁, 贾长青, 韩长旭, 等. 生长分化因子 5 诱导兔脂肪干细胞成软骨细胞分化的实验研究. 中国修复重建外科杂志, 2009, 23(4): 483-489.
10. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 2001, 7(2): 211-228.
11. 史琳丽, 杨向群. 脂肪组织来源干细胞的分化潜能和应用. 中国修复重建外科杂志, 2012, 26(8): 1007-1011.
12. Erickson GR, Gimble JM, Franklin DM, et al. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun, 2002, 290(2): 763-769.
13. Messai H, Duchossoy Y, Khatib AM, et al. Articular chondrocytes from aging rats respond poorly to insulin-like growth factor-1: an altered signaling pathway. Mech Ageing Dev, 2000, 115(1-2): 21-37.
14. 杨强, 丁晓明, 徐宝山, 等. 取向性丝素蛋白支架复合脂肪干细胞体外构建组织工程软骨. 中华骨科杂志, 2015, 35(12): 1235-1242.
15. An C, Cheng Y, Yuan Q, et al. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng, 2010, 38(4): 1647-1654.
16. Zheng W, Gao X, Gu Q, et al. Antitumor activity of daphnodorins from Daphne genkwa roots. Int Immunopharmacol, 2007, 7(2): 128-134.
17. Prince M, Li Y, Childers A, et al. Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice. Toxicol Lett, 2009, 185(3): 180-186.
18. Cottiglia F, Loy G, Garau D, et al. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium L. Phytomedicine, 2001, 8(4): 302-305.
19. 邹文玮, 舒奇, 傅颖媛, 等. 瑞香素毒理学实验研究. 中成药, 2013, 35(5): 908-911.
20. Gao Q, Shan J, Di L, et al. Therapeutic effects of daphnetin on adjuvant-induced arthritic rats. J Ethnopharmacol, 2008, 120(2): 259-263.
21. Tang DZ, Hou W, Zhou Q, et al. Osthole stimulates osteoblast differentiation and bone formation by activation of beta-catenin-BMP signaling. J Bone Miner Res, 2010, 25(6): 1234-1245.
22. Liu S, Shao Y, Lin Q, et al. 7, 8-Dihydroxy coumarin promotes chondrogenic differentiation of adipose-derived mesenchymal stem cells. J Int Med Res, 2013, 41(1): 82-96.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of daphnetin combined with insulin-like growth factor 1 on chondrogenic differentiation of adipose-derived mesenchymal stem cells in rats

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