BMP-2 聯合低氧環境誘導BMSCs 向軟骨表型分化的研究_《中國修復重建外科雜志》

作者：

廖新根 ^1,2 , 吳梨華 ² , 付明福 ^1,2 , 何丁文 ^1,2 , 顧玉榮 ¹ , 陳偉才 ¹ , 殷明 ¹

1 南昌大學第二附屬醫院骨一科（南昌，330006）;;
2 南昌大學醫學院研究生部;

關鍵詞：

BMP-2 低氧 BMSCs 軟骨表型分化大鼠

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目的探討BMP-2 聯合低氧環境誘導BMSCs 向軟骨表型分化的可行性，并進一步研究其生物學機制。方法取4 周齡健康清潔級雌性SD 大鼠骨髓采用貼壁法體外培養BMSCs，取第2 代細胞根據培養條件不同分為4 組：常氧對照組（A 組）、常氧加BMP-2 誘導液組（B 組）、低氧（O2 濃度3%）對照組（C 組）和低氧加BMP-2誘導液組（D 組）。倒置相差顯微鏡下觀察細胞形態變化，培養7、14、21 d 阿利新藍染色檢測各組軟骨基質糖胺聚糖（glycosaminoglycans，GAG）分泌水平，21 d 時Western blot 檢測細胞內Ⅱ型膠原和低氧誘導因子1α（hypoxiainducible factor 1α，HIF-1α）蛋白表達水平，RT-PCR檢測成軟骨、成骨以及低氧相關基因表達水平。結果誘導培養21 d，D 組細胞變為類圓形，細胞密度降低，細胞周邊呈陷窩樣，基質包裹細胞；A、B、C 組均未見上述典型變化。D 組阿利新藍染色明顯較其他組深，并隨誘導時間延長藍染加深，21 d 時出現成片深染藍色，其他組各時間點僅見散在少量的淡染藍色。Western blot 檢測D 組細胞內Ⅱ型膠原蛋白表達水平較其他組顯著增高，C、D 組HIF-1α 蛋白表達水平較A、B 組顯著增高，差異均有統計學意義（P lt; 0.05）。RT-PCR 檢測D 組成軟骨分化相關指標Ⅱ型膠原α1（collagen Ⅱ α1，COL2 α1）、聚集蛋白聚糖表達最高，而B 組成骨分化相關指標COL1 α1、ALP、Runt 相關轉錄因子2 表達水平最高，C、D 組低氧相關指標HIF-1α 較A、B 組顯著增強，差異均有統計學意義（P lt; 0.05）。結論 BMP-2 聯合低氧（O2 濃度3%）環境可以誘導大鼠BMSCs 向軟骨分化，并抑制其成骨分化，HIF-1α 可能是參與促軟骨生成過程中的一個重要信號分子。

引用本文： 廖新根,吳梨華,付明福,何丁文,顧玉榮,陳偉才,殷明. BMP-2 聯合低氧環境誘導BMSCs 向軟骨表型分化的研究. 中國修復重建外科雜志, 2012, 26(6): 743-748. doi: 復制

1.	Toh WS, Yang Z, Liu H, et al. Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells, 2007, 25(4): 950-960.
2.	Solorio LD, Vieregge EL, Dhami CD, et al. Engineered cartilage via self-assembled hmsc sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-beta1. J Control Release, 2012, 158(2): 224-232.
3.	Dani?ovi? L, Varga I, Polák S. Growth factors and chondrogenic differentiation of mesenchymal stem cells. Tissue Cell, 2012, 44(2): 69-73.
4.	Adams CS, Shapiro IM. The fate of the terminally differentiated chondrocyte: Evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev Oral Biol Med, 2002, 13(6): 465-473.
5.	Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood, 2003, 102(10): 3483-3493.
6.	Rocha B, Fernández-Puente P, Mateos J, et al. A quantitative proteomics approach for studying the chondrogenic differentiation process of mesenchymal stem cells. Osteoarthritis and Cartilage, 2011, 19S1: S46-S47.
7.	劉哲, 李士勇, 宋玉林, 等. 綠原酸對缺氧環境下干細胞來源軟骨樣細胞凋亡的影響. 中國藥理學通報, 2011, 27(2): 206-210.
8.	Steinert AF, Proffen B, Kunz M, et al. Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer. Arthritis Res Ther, 2009, 11(5): R148.
9.	Rosen V. BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev, 2009, 20(5-6): 475-480.
10.	Wang DW, Fermor B, Gimble JM, et al. Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells. J Cell Physiol, 2005, 204(1): 184-191.
11.	Markway BD, Tan GK, Brooke G, et al. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplant, 2010, 19(1): 29-42.
12.	Kanichai M, Ferguson D, Prendergast PJ, et al. Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: A role for akt and hypoxia-inducible factor (hif)-1alpha. J Cell Physiol, 2008, 216(3): 708-715.
13.	Kay A, Richardson J, Forsyth NR. Physiological normoxia and chondrogenic potential of chondrocytes. Front Biosci (Elite Ed), 2011, 3: 1365-1374.
14.	D’Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human miami cells. Bone, 2006, 39(3): 513-522.
15.	Merceron C, Vinatier C, Portron S, et al. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol, 2010, 298(2): C355-364.
16.	Singh M, Pierpoint M, Mikos AG, et al. Chondrogenic differentiation of neonatal human dermal fibroblasts encapsulated in alginate beads with hydrostatic compression under hypoxic conditions in the presence of bone morphogenetic protein-2. J Biomed Mater Res A, 2011, 98(3): 412-424.
17.	Pelaez D, Arita N, Cheung HS. Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression. Biochem Biophys Res Commun, 2012, 417(4): 1286-1291.
18.	Sheehy EJ, Buckley CT, Kelly DJ. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells. Biochem Biophys Res Commun, 2012, 417(1): 305-310.
19.	Diaz-Prado S, Muiños-Lopez E, Hermida-Gómez T, et al. Chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCS) grown on collagen porous scaffolds. Osteoarthritis and Cartilage, 2011, 19S1: S221.
20.	Ronzière MC, Perrier E, Mallein-Gerin F, et al. Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells. Biomed Mater Eng, 2010, 20(3): 145-158.
21.	Bosetti M, Boccafoschi F, Leigheb M, et al. Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering. J Tissue Eng Regen Med, 2011, 6(3): 205-213.

1. Toh WS, Yang Z, Liu H, et al. Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells, 2007, 25(4): 950-960.
2. Solorio LD, Vieregge EL, Dhami CD, et al. Engineered cartilage via self-assembled hmsc sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-beta1. J Control Release, 2012, 158(2): 224-232.
3. Dani?ovi? L, Varga I, Polák S. Growth factors and chondrogenic differentiation of mesenchymal stem cells. Tissue Cell, 2012, 44(2): 69-73.
4. Adams CS, Shapiro IM. The fate of the terminally differentiated chondrocyte: Evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev Oral Biol Med, 2002, 13(6): 465-473.
5. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood, 2003, 102(10): 3483-3493.
6. Rocha B, Fernández-Puente P, Mateos J, et al. A quantitative proteomics approach for studying the chondrogenic differentiation process of mesenchymal stem cells. Osteoarthritis and Cartilage, 2011, 19S1: S46-S47.
7. 劉哲, 李士勇, 宋玉林, 等. 綠原酸對缺氧環境下干細胞來源軟骨樣細胞凋亡的影響. 中國藥理學通報, 2011, 27(2): 206-210.
8. Steinert AF, Proffen B, Kunz M, et al. Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer. Arthritis Res Ther, 2009, 11(5): R148.
9. Rosen V. BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev, 2009, 20(5-6): 475-480.
10. Wang DW, Fermor B, Gimble JM, et al. Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells. J Cell Physiol, 2005, 204(1): 184-191.
11. Markway BD, Tan GK, Brooke G, et al. Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. Cell Transplant, 2010, 19(1): 29-42.
12. Kanichai M, Ferguson D, Prendergast PJ, et al. Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: A role for akt and hypoxia-inducible factor (hif)-1alpha. J Cell Physiol, 2008, 216(3): 708-715.
13. Kay A, Richardson J, Forsyth NR. Physiological normoxia and chondrogenic potential of chondrocytes. Front Biosci (Elite Ed), 2011, 3: 1365-1374.
14. D’Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human miami cells. Bone, 2006, 39(3): 513-522.
15. Merceron C, Vinatier C, Portron S, et al. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol, 2010, 298(2): C355-364.
16. Singh M, Pierpoint M, Mikos AG, et al. Chondrogenic differentiation of neonatal human dermal fibroblasts encapsulated in alginate beads with hydrostatic compression under hypoxic conditions in the presence of bone morphogenetic protein-2. J Biomed Mater Res A, 2011, 98(3): 412-424.
17. Pelaez D, Arita N, Cheung HS. Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression. Biochem Biophys Res Commun, 2012, 417(4): 1286-1291.
18. Sheehy EJ, Buckley CT, Kelly DJ. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells. Biochem Biophys Res Commun, 2012, 417(1): 305-310.
19. Diaz-Prado S, Muiños-Lopez E, Hermida-Gómez T, et al. Chondrogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCS) grown on collagen porous scaffolds. Osteoarthritis and Cartilage, 2011, 19S1: S221.
20. Ronzière MC, Perrier E, Mallein-Gerin F, et al. Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells. Biomed Mater Eng, 2010, 20(3): 145-158.
21. Bosetti M, Boccafoschi F, Leigheb M, et al. Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering. J Tissue Eng Regen Med, 2011, 6(3): 205-213.

《中國修復重建外科雜志》

BMP-2 聯合低氧環境誘導BMSCs 向軟骨表型分化的研究

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《中國修復重建外科雜志》

BMP-2 聯合低氧環境誘導BMSCs 向軟骨表型分化的研究

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Content

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