不同时期bFGF或副甲状腺激素相关肽对TGF-β1诱导的兔BMSCs向软骨细胞分化的影响_《中国修复重建外科杂志》

作者：

刘印 , 贺利平 , 田京

南方医科大学珠江医院骨科（广州，510282）;

关键词：

软骨组织工程 BMSCs bFGF 副甲状腺激素相关肽成软骨分化兔

DOI：

10.7507/1002-1892.20130044

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的探讨bFGF和副甲状腺激素相关肽（parathyroid hormone-related protein，PTHrP）对TGF-β1诱导兔BMSCs向软骨细胞分化的影响。方法2月龄健康日本大耳白兔3只，雌雄不限，体重1.6～2.1 kg；取兔胫骨骨髓分离培养BMSCs。取第3代细胞行团块状立体培养，并按照不同诱导条件分为TGF-β1组（A组）、TGF-β1/bFGF组（B组）、TGF-β1/21 d bFGF组（C组）、TGF-β1/PTHrP组（D组）、TGF-β1/21d PTHrP组（E组）。于团块状立体培养开始时，各组均加入10 ng/mL TGF-β1；B、D组同时加入10 ng/mL bFGF或10 ng/mL PTHrP；C、E组于培养21 d时对应加入10 ng/ mL bFGF或10 ng/mL PTHrP。培养后1、2、3、4、5、6周检测各组BMSCs向软骨细胞分化的标志性基因Ⅰ型胶原（collagen type Ⅰ，ColⅠ）、ColⅡ、ColⅩ和基质金属蛋白酶（matrix metalloproteinases，MMP）13的表达，1、2、3、4、6周检测ALP活性，6周时行1，9二甲基亚甲蓝（1，9-dimethylmethylene blue，DMMB）染色观察细胞外基质分泌情况。结果RT-PCR检测示，3周后C、E组ColⅠ基因表达呈显著下降趋势，4、5周时A组显著高于C、E组（P lt; 0.05），3～6周A组显著高于B、D组（P lt; 0.05）；B、C组3、4周时及D、E组3周时比较，差异有统计学意义（P lt; 0.05）。C、E组3周后ColⅡ、ColⅩ基因表达均逐渐下降，4～6周时显著低于A组（P lt; 0.05）；B、D组各时间点两基因表达均未见显著升高，与A组比较差异均有统计学意义（P lt; 0.05）。A组各时间点MMP-13均未见明显表达，3周时B组与A、C组比较差异有统计学意义（P lt; 0.05），D组显著高于A、E组（P lt; 0.05）。随着时间延长A组ALP活性逐渐升高，4周后C、E组显著下降，均低于A组（P lt; 0.05）；B、C组间及D、E组间比较，仅在2、3周时差异有统计学意义（P lt; 0.05）。DMMB染色显示6周时A组软骨陷窝明显，其余各组软骨陷窝明显较少。结论bFGF和PHTrP能通过改变软骨细胞外基质的合成和分解，抑制TGF-β1诱导的兔BMSCs向软骨细胞分化。这种抑制作用不仅通过抑制ColⅩ基因表达而实现，可能还通过抑制其他软骨分化相关蛋白实现。

引用本文： 刘印,贺利平,田京. 不同时期bFGF或副甲状腺激素相关肽对TGF-β1诱导的兔BMSCs向软骨细胞分化的影响. 中国修复重建外科杂志, 2013, 27(2): 199-206. doi: 10.7507/1002-1892.20130044 复制

1.	Zheng YH, Su K, Jian YT, et al. Basic fibroblast growth factor enhances osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells in coral scaffold constructs. J Tissue Eng Regen Med, 2011, 5(7): 540-550.
2.	Kim JH, Lee MC, Seong SC, et al. Enhanced proliferation and chondrogenic differentiation of human synovium-derived stem cells expanded with basic fibroblast growth factor. Tissue Eng Part A, 2011, 17(7-8): 991-1002.
3.	Lee S, Kim JH, Jo CH, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Eng Part A, 2009, 15(11): 3401-3415.
4.	Park KH, Na K. Effect of growth factors on chondrogenic differentiation of rabbit mesenchymal cells embedded in injectable hydrogels. J Biosci Bioeng, 2008, 106(1): 74-79.
5.	Yang KG, Saris DB, Geuze RE, et al. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng, 2006, 12(9): 2435-2447.
6.	Bosnakovski D, Mizuno M, Kim G, et al. Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system. Jpn J Vet Res, 2006, 53(3-4): 127-139.
7.	Mizuta H, Kudo S, Nakamura E, et al. Expression of the PTH/PTHrP receptor in chondrogenic cells during the repair of full-thickness defects of articular cartilage. Osteoarthritis Cartilage, 2006, 14(9): 944-952.
8.	Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood, 2008, 112(2): 295-307.
9.	Ito T, Sawada R, Fujiwara Y, et al. FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling. Cytotechnology, 2008, 56(1): 1-7.
10.	Harrington EK, Roddy GW, West R, et al. Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation. Anat Rec (Hoboken), 2007, 290(2): 155-167.
11.	Amizuka N, Davidson D, Liu H, et al. Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone, 2004, 34(1): 13-25.
12.	Hankemeier S, Keus M, Zeichen J, et al. Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng, 2005, 11(1-2): 41-49.
13.	Dickhut A, Gottwald E, Steck E, et al. Chondrogenesis of mesenchymal stem cells in gel-like biomaterials in vitro and in vivo. Front Biosci, 2008, 13: 4517-4528.
14.	Varas L, Ohlsson LB, Honeth G, et al. Alpha10 integrin expression is up-regulated on fibroblast growth factor-2-treated mesenchymal stem cells with improved chondrogenic differentiation potential. Stem Cells Dev, 2007, 16(6): 965-978.
15.	Iwamoto M, Jikko A, Murakami H, et al. Changes in parathyroid hormone receptors during chondrocyte cytodifferentiation. J Biol Chem, 1994, 269(25): 17245-17251.
16.	Kafienah W, Mistry S, Dickinson SC, et al. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis Rheum, 2007, 56(1): 177-187.
17.	Kim YJ, Kim HJ, Im GI. PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs. Biochem Biophys Res Commun, 2008, 373(1): 104-108.
18.	晏杰, 刘玲蓉, 张其清. 诱导骨髓间充质干细胞向软骨细胞分化的体外研究. 中国修复重建外科杂志, 2006, 20(11): 1114-1118.

1. Zheng YH, Su K, Jian YT, et al. Basic fibroblast growth factor enhances osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells in coral scaffold constructs. J Tissue Eng Regen Med, 2011, 5(7): 540-550.
2. Kim JH, Lee MC, Seong SC, et al. Enhanced proliferation and chondrogenic differentiation of human synovium-derived stem cells expanded with basic fibroblast growth factor. Tissue Eng Part A, 2011, 17(7-8): 991-1002.
3. Lee S, Kim JH, Jo CH, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Eng Part A, 2009, 15(11): 3401-3415.
4. Park KH, Na K. Effect of growth factors on chondrogenic differentiation of rabbit mesenchymal cells embedded in injectable hydrogels. J Biosci Bioeng, 2008, 106(1): 74-79.
5. Yang KG, Saris DB, Geuze RE, et al. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng, 2006, 12(9): 2435-2447.
6. Bosnakovski D, Mizuno M, Kim G, et al. Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system. Jpn J Vet Res, 2006, 53(3-4): 127-139.
7. Mizuta H, Kudo S, Nakamura E, et al. Expression of the PTH/PTHrP receptor in chondrogenic cells during the repair of full-thickness defects of articular cartilage. Osteoarthritis Cartilage, 2006, 14(9): 944-952.
8. Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood, 2008, 112(2): 295-307.
9. Ito T, Sawada R, Fujiwara Y, et al. FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling. Cytotechnology, 2008, 56(1): 1-7.
10. Harrington EK, Roddy GW, West R, et al. Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation. Anat Rec (Hoboken), 2007, 290(2): 155-167.
11. Amizuka N, Davidson D, Liu H, et al. Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone, 2004, 34(1): 13-25.
12. Hankemeier S, Keus M, Zeichen J, et al. Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng, 2005, 11(1-2): 41-49.
13. Dickhut A, Gottwald E, Steck E, et al. Chondrogenesis of mesenchymal stem cells in gel-like biomaterials in vitro and in vivo. Front Biosci, 2008, 13: 4517-4528.
14. Varas L, Ohlsson LB, Honeth G, et al. Alpha10 integrin expression is up-regulated on fibroblast growth factor-2-treated mesenchymal stem cells with improved chondrogenic differentiation potential. Stem Cells Dev, 2007, 16(6): 965-978.
15. Iwamoto M, Jikko A, Murakami H, et al. Changes in parathyroid hormone receptors during chondrocyte cytodifferentiation. J Biol Chem, 1994, 269(25): 17245-17251.
16. Kafienah W, Mistry S, Dickinson SC, et al. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis Rheum, 2007, 56(1): 177-187.
17. Kim YJ, Kim HJ, Im GI. PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs. Biochem Biophys Res Commun, 2008, 373(1): 104-108.
18. 晏杰, 刘玲蓉, 张其清. 诱导骨髓间充质干细胞向软骨细胞分化的体外研究. 中国修复重建外科杂志, 2006, 20(11): 1114-1118.

《中国修复重建外科杂志》

不同时期bFGF或副甲状腺激素相关肽对TGF-β1诱导的兔BMSCs向软骨细胞分化的影响

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《中国修复重建外科杂志》

不同时期bFGF或副甲状腺激素相关肽对TGF-β1诱导的兔BMSCs向软骨细胞分化的影响

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