不同時期bFGF或副甲狀腺激素相關肽對TGF-β1誘導的兔BMSCs向軟骨細胞分化的影響_《中國修復重建外科雜志》

作者：

劉印 , 賀利平 , 田京

南方醫科大學珠江醫院骨科（廣州，510282）;

關鍵詞：

軟骨組織工程 BMSCs bFGF 副甲狀腺激素相關肽成軟骨分化兔

DOI：

10.7507/1002-1892.20130044

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目的探討bFGF和副甲狀腺激素相關肽（parathyroid hormone-related protein，PTHrP）對TGF-β1誘導兔BMSCs向軟骨細胞分化的影響。方法2月齡健康日本大耳白兔3只，雌雄不限，體重1.6～2.1 kg；取兔脛骨骨髓分離培養BMSCs。取第3代細胞行團塊狀立體培養，并按照不同誘導條件分為TGF-β1組（A組）、TGF-β1/bFGF組（B組）、TGF-β1/21 d bFGF組（C組）、TGF-β1/PTHrP組（D組）、TGF-β1/21d PTHrP組（E組）。于團塊狀立體培養開始時，各組均加入10 ng/mL TGF-β1；B、D組同時加入10 ng/mL bFGF或10 ng/mL PTHrP；C、E組于培養21 d時對應加入10 ng/ mL bFGF或10 ng/mL PTHrP。培養后1、2、3、4、5、6周檢測各組BMSCs向軟骨細胞分化的標志性基因Ⅰ型膠原（collagen type Ⅰ，ColⅠ）、ColⅡ、ColⅩ和基質金屬蛋白酶（matrix metalloproteinases，MMP）13的表達，1、2、3、4、6周檢測ALP活性，6周時行1，9二甲基亞甲藍（1，9-dimethylmethylene blue，DMMB）染色觀察細胞外基質分泌情況。結果RT-PCR檢測示，3周后C、E組ColⅠ基因表達呈顯著下降趨勢，4、5周時A組顯著高于C、E組（P lt; 0.05），3～6周A組顯著高于B、D組（P lt; 0.05）；B、C組3、4周時及D、E組3周時比較，差異有統計學意義（P lt; 0.05）。C、E組3周后ColⅡ、ColⅩ基因表達均逐漸下降，4～6周時顯著低于A組（P lt; 0.05）；B、D組各時間點兩基因表達均未見顯著升高，與A組比較差異均有統計學意義（P lt; 0.05）。A組各時間點MMP-13均未見明顯表達，3周時B組與A、C組比較差異有統計學意義（P lt; 0.05），D組顯著高于A、E組（P lt; 0.05）。隨著時間延長A組ALP活性逐漸升高，4周后C、E組顯著下降，均低于A組（P lt; 0.05）；B、C組間及D、E組間比較，僅在2、3周時差異有統計學意義（P lt; 0.05）。DMMB染色顯示6周時A組軟骨陷窩明顯，其余各組軟骨陷窩明顯較少。結論bFGF和PHTrP能通過改變軟骨細胞外基質的合成和分解，抑制TGF-β1誘導的兔BMSCs向軟骨細胞分化。這種抑制作用不僅通過抑制ColⅩ基因表達而實現，可能還通過抑制其他軟骨分化相關蛋白實現。

引用本文： 劉印,賀利平,田京. 不同時期bFGF或副甲狀腺激素相關肽對TGF-β1誘導的兔BMSCs向軟骨細胞分化的影響. 中國修復重建外科雜志, 2013, 27(2): 199-206. doi: 10.7507/1002-1892.20130044 復制

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, 劉玲蓉, 張其清. 誘導骨髓間充質干細胞向軟骨細胞分化的體外研究. 中國修復重建外科雜志, 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, 劉玲蓉, 張其清. 誘導骨髓間充質干細胞向軟骨細胞分化的體外研究. 中國修復重建外科雜志, 2006, 20(11): 1114-1118.

《中國修復重建外科雜志》

不同時期bFGF或副甲狀腺激素相關肽對TGF-β1誘導的兔BMSCs向軟骨細胞分化的影響

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《中國修復重建外科雜志》

不同時期bFGF或副甲狀腺激素相關肽對TGF-β1誘導的兔BMSCs向軟骨細胞分化的影響

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