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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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.	宋克東, 楊延飛, 李文芳, 等. ADSCs-殼聚糖/明膠水凝膠工程化軟骨的三維動態構建. 功能材料, 2015, 46(15): 15021-15030.
4.	張勇, 陳曉菊, 王燕杰, 等. 兔脂肪干細胞直接修復陳舊性全層兔膝關節軟骨缺損. 臨床骨科雜志, 2015, 18(4): 492-497.
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6.	趙明璨, 劉暢. 脂肪干細胞在軟骨組織工程中的研究進展. 中國生物醫學工程學報, 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.	朱國華, 齊新生. 關節軟骨缺損修復的實驗與臨床. 中國骨傷, 2004, 17(5): 318-320.
9.	劉振寧, 賈長青, 韓長旭, 等. 生長分化因子 5 誘導兔脂肪干細胞成軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 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.	史琳麗, 楊向群. 脂肪組織來源干細胞的分化潛能和應用. 中國修復重建外科雜志, 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.	楊強, 丁曉明, 徐寶山, 等. 取向性絲素蛋白支架復合脂肪干細胞體外構建組織工程軟骨. 中華骨科雜志, 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.	鄒文瑋, 舒奇, 傅穎媛, 等. 瑞香素毒理學實驗研究. 中成藥, 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. 宋克東, 楊延飛, 李文芳, 等. ADSCs-殼聚糖/明膠水凝膠工程化軟骨的三維動態構建. 功能材料, 2015, 46(15): 15021-15030.
4. 張勇, 陳曉菊, 王燕杰, 等. 兔脂肪干細胞直接修復陳舊性全層兔膝關節軟骨缺損. 臨床骨科雜志, 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. 趙明璨, 劉暢. 脂肪干細胞在軟骨組織工程中的研究進展. 中國生物醫學工程學報, 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. 朱國華, 齊新生. 關節軟骨缺損修復的實驗與臨床. 中國骨傷, 2004, 17(5): 318-320.
9. 劉振寧, 賈長青, 韓長旭, 等. 生長分化因子 5 誘導兔脂肪干細胞成軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 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. 史琳麗, 楊向群. 脂肪組織來源干細胞的分化潛能和應用. 中國修復重建外科雜志, 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. 楊強, 丁曉明, 徐寶山, 等. 取向性絲素蛋白支架復合脂肪干細胞體外構建組織工程軟骨. 中華骨科雜志, 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. 鄒文瑋, 舒奇, 傅穎媛, 等. 瑞香素毒理學實驗研究. 中成藥, 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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