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(xué)第二附屬醫(yī)院骨一科（南昌，330006）;;
  • 2 南昌大學(xué)醫(yī)學(xué)院研究生部;

目的 探討B(tài)MP-2 聯(lián)合低氧環(huán)境誘導(dǎo)BMSCs 向軟骨表型分化的可行性，并進(jìn)一步研究其生物學(xué)機(jī)制。 方法 取4 周齡健康清潔級(jí)雌性SD 大鼠骨髓采用貼壁法體外培養(yǎng)BMSCs，取第2 代細(xì)胞根據(jù)培養(yǎng)條件不同分為4 組：常氧對(duì)照組（A 組）、常氧加BMP-2 誘導(dǎo)液組（B 組）、低氧（O2 濃度3%）對(duì)照組（C 組）和低氧加BMP-2誘導(dǎo)液組（D 組）。倒置相差顯微鏡下觀(guān)察細(xì)胞形態(tài)變化，培養(yǎng)7、14、21 d 阿利新藍(lán)染色檢測(cè)各組軟骨 基質(zhì)糖胺聚糖（glycosaminoglycans，GAG）分泌水平，21 d 時(shí)Western blot 檢測(cè)細(xì)胞內(nèi)Ⅱ型膠原和低氧誘導(dǎo)因子1α（hypoxiainducible factor 1α，HIF-1α）蛋白表達(dá)水平，RT-PCR檢測(cè)成軟骨、成骨以及低氧相關(guān)基因表達(dá)水平。 結(jié)果 誘導(dǎo)培養(yǎng)21 d，D 組細(xì)胞變?yōu)轭?lèi)圓形，細(xì)胞密度降低，細(xì)胞周邊呈陷窩樣，基質(zhì)包裹細(xì)胞；A、B、C 組均未見(jiàn)上述典型變化。D 組阿利新藍(lán)染色明顯較其他組深，并隨誘導(dǎo)時(shí)間延長(zhǎng)藍(lán)染加深，21 d 時(shí)出現(xiàn)成片深染藍(lán)色，其他組各時(shí)間點(diǎn)僅見(jiàn)散在少量的淡染藍(lán)色。Western blot 檢測(cè)D 組細(xì)胞內(nèi)Ⅱ型膠原蛋白表達(dá)水平較其他組顯著增高，C、D 組HIF-1α 蛋白表達(dá)水平較A、B 組顯著增高，差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。RT-PCR 檢測(cè)D 組成軟骨分化相關(guān)指標(biāo)Ⅱ型膠原α1（collagen Ⅱ α1，COL2 α1）、聚集蛋白聚糖表達(dá)最高，而B(niǎo) 組成骨分化相關(guān)指標(biāo)COL1 α1、ALP、Runt 相關(guān)轉(zhuǎn)錄因子2 表達(dá)水平最高，C、D 組低氧相關(guān)指標(biāo)HIF-1α 較A、B 組顯著增強(qiáng)，差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。 結(jié)論 BMP-2 聯(lián)合低氧（O2 濃度3%）環(huán)境可以誘導(dǎo)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(guò)程中的一個(gè)重要信號(hào)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(lián)合低氧環(huán)境誘導(dǎo)BMSCs 向軟骨表型分化的研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(6): 743-748. doi: 復(fù)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(duì)缺氧環(huán)境下干細(xì)胞來(lái)源軟骨樣細(xì)胞凋亡的影響. 中國(guó)藥理學(xué)通報(bào), 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. 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. 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. 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. 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. 5. Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood, 2003, 102(10): 3483-3493.
  6. 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. 7. 劉哲, 李士勇, 宋玉林, 等. 綠原酸對(duì)缺氧環(huán)境下干細(xì)胞來(lái)源軟骨樣細(xì)胞凋亡的影響. 中國(guó)藥理學(xué)通報(bào), 2011, 27(2): 206-210.
  8. 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. 9. Rosen V. BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev, 2009, 20(5-6): 475-480.
  10. 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. 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. 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. 13. Kay A, Richardson J, Forsyth NR. Physiological normoxia and chondrogenic potential of chondrocytes. Front Biosci (Elite Ed), 2011, 3: 1365-1374.
  14. 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. 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. 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. 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. 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. 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. 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. 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.