• 解放軍總醫(yī)院（北京，100853）1 骨科，3 急救部;;
  • 2 軍事醫(yī)學(xué)科學(xué)院輻射醫(yī)學(xué)研究所;

目的 明確萎縮性骨不連組織中表達(dá)上調(diào)的數(shù)種微小RNA（micro RNA，miRNA）與其相應(yīng)靶基因mRNA、蛋白在hBMSCs 成骨分化過(guò)程中的表達(dá)變化趨勢(shì)和生物學(xué)功能。 方法 取自體髂骨植骨手術(shù)患者的髂骨骨髓血，采用密度梯度離心法分離培養(yǎng)hBMSCs。取第4 代hBMSCs 以成骨誘導(dǎo)培養(yǎng)液誘導(dǎo)成骨分化，分別提取0、12 h，1、2、4、7、14 d 時(shí)的細(xì)胞總RNA 和蛋白，進(jìn)行miRNA 的實(shí)時(shí)定量PCR（quantitative real-time PCR，qRT-PCR）、相應(yīng)靶基因mRNA 的qRT-PCR 和蛋白Western blot 檢測(cè)。 結(jié)果 誘導(dǎo)hBMSCs 成骨分化時(shí)，成骨性靶基因堿性磷酸酶（alkaline phosphatase liver/bone/kidney，ALPL）、PDGF-α 多肽（PDGF-α polypeptide，PDGF-A）和BMP-2 的mRNA 和蛋白表達(dá)在多數(shù)時(shí)間點(diǎn)同對(duì)照（0 h）相比增加（BMP-2 在12 h 和1 d 時(shí)下降），1 ～ 7 d 變化最為顯著。不同時(shí)間點(diǎn)的miRNA、靶基因mRNA 和蛋白表達(dá)水平存在差異，其中hsa-miRNA-149 和hsa-miRNA-654-5p miRNA 含量的變化趨勢(shì)與各自靶基因ALPL 和BMP-2 的mRNA 及蛋白表達(dá)水平總體上成負(fù)相關(guān)（P  lt; 0.05），hsa-miRNA-221 與其靶基因PDGF-A 的變化趨勢(shì)無(wú)明顯負(fù)相關(guān)關(guān)系（P  gt; 0.05）。 結(jié)論 誘導(dǎo)hBMSCs 成骨分化過(guò)程中，hsa-miRNA-149 和hsa-miRNA-654-5p 對(duì)其相應(yīng)靶基因ALPL 和BMP-2 的mRNA 及蛋白存在密切調(diào)控關(guān)系。

引用本文： 魏均強(qiáng) ,張伯勛,鄭曉飛,陳華,田學(xué)忠,唐佩福,宋青,黎檀實(shí). 誘導(dǎo)hBMSCs 成骨分化中微小RNA 和靶基因表達(dá)水平的變化. 中國(guó)修復(fù)重建外科雜志, 2012, 26(4): 483-487. doi: 復(fù)制

1. 魏均強(qiáng). miRNA在萎縮性骨不連中的調(diào)控作用及其分子生物學(xué)機(jī)制. 北京: 中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院, 2011.
2. Service RF. Tissue engineers build new bone. Science, 2000, 289(5484): 1498-1500.
3. Satija NK, Gurudutta GU, Sharma S, et al. Mesenchymal stem cells: molecular targets for tissue engineering. Stem Cells Dev, 2007, 16(1): 7-23.
4. Xie C, Reynolds D, Awad H, et al. Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering. Tissue Eng, 2007, 13(3): 435-445.
5. Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.
6. Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103.
7. 胡煒, 俞興, 徐林. 骨髓基質(zhì)細(xì)胞誘導(dǎo)分化成骨方法及相關(guān)研究進(jìn)展. 中國(guó)組織工程研究與臨床康復(fù), 2009, 13(1): 169-172.
8. Niemeyer P, Krause U, Punzel M, et al. Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen. Z Orthop Ihre Grenzgeb, 2003, 141(6): 712-717.
9. Taira M, Nakao H, Takahashi J, et al. Effects of two vitamins, two growth factors and dexamethasone on the proliferation of rat bone marrow stromal cells and osteoblastic MC3T3-E1cells. J Oral Rehabil, 2003, 30(7): 697-701.
10. Rodríguez JP, González M, Ríos S, et al. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem, 2004, 93(4): 721-731.
11. Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterizations of osteoblastic differentiation of stromalcell line ST2 that is induced by ascorbic acid. Am J Physiol, 1999, 277(1 Pt 1): C132-138.
12. Egusa H, Iida K, Kobayashi M, et al. Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells. Tissue Eng, 2007, 13(10): 2589-2600.
13. Timmer NM, van Horssen J, Otte-Holler I, et al. Amyloid beta induces cellular relocalization and production of agrin and glypican-1. Brain Res, 2009, 1260: 38-46.
14. Oreffo RO, Cooper C, Mason C, et al. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev, 2005, 1(2): 169-178.
15. Kimelman N, Pelled G, Gazit Z, et al. Applications of gene therapy and adult stem cells in bone bioengineering. Regen Med, 2006, 1(4): 549-561.
16. Gugala Z, Olmsted-Davis EA, Gannon FH, et al. Osteo-induction by ex vivo adenovirus-mediated BMP-2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.
17. Gugala Z, Davis AR, Fouletier-Dilling CM, et al. Adenovirus BMP-2 induced osteogenesis in combination with collagen carriers. Biomaterials, 2007, 28(30): 4469-4479.
18. Sipos W, Pietschmann P, Rauner M. Strategies for novel therapeutic approaches targeting cytokines and signaling pathways of osteoclasto- and osteoblastogenesis in the fight against immune-mediated bone and joint diseases. Curr Med Chem, 2008, 15(2): 127-136.
19. Alcaraz MJ, Megias J, Garcia-Arnandis I, et al. New molecular targets for the treatment of osteoarthritis. Biochem Pharmacol, 2010, 80(1): 13-21.
20. Fulci V, Scappucci G, Sebastiani GD, et al. miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol, 2010, 71(2): 206-211.
21. Luo X, Tsai LM, Shen N, et al. Evidence for microRNA-mediated regulation in rheumatic diseases. Ann Rheum Dis, 2010, 69 Suppl 1: i30-36.
22. Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.
23. Babkina IV, Osipov DA, Solovyov YN, et al. Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary osteosarcomas. Bull Exp Biol Med, 2009, 148(2): 246-249.
  1. 1. 魏均強(qiáng). miRNA在萎縮性骨不連中的調(diào)控作用及其分子生物學(xué)機(jī)制. 北京: 中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院, 2011.
  2. 2. Service RF. Tissue engineers build new bone. Science, 2000, 289(5484): 1498-1500.
  3. 3. Satija NK, Gurudutta GU, Sharma S, et al. Mesenchymal stem cells: molecular targets for tissue engineering. Stem Cells Dev, 2007, 16(1): 7-23.
  4. 4. Xie C, Reynolds D, Awad H, et al. Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering. Tissue Eng, 2007, 13(3): 435-445.
  5. 5. Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.
  6. 6. Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103.
  7. 7. 胡煒, 俞興, 徐林. 骨髓基質(zhì)細(xì)胞誘導(dǎo)分化成骨方法及相關(guān)研究進(jìn)展. 中國(guó)組織工程研究與臨床康復(fù), 2009, 13(1): 169-172.
  8. 8. Niemeyer P, Krause U, Punzel M, et al. Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen. Z Orthop Ihre Grenzgeb, 2003, 141(6): 712-717.
  9. 9. Taira M, Nakao H, Takahashi J, et al. Effects of two vitamins, two growth factors and dexamethasone on the proliferation of rat bone marrow stromal cells and osteoblastic MC3T3-E1cells. J Oral Rehabil, 2003, 30(7): 697-701.
  10. 10. Rodríguez JP, González M, Ríos S, et al. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem, 2004, 93(4): 721-731.
  11. 11. Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterizations of osteoblastic differentiation of stromalcell line ST2 that is induced by ascorbic acid. Am J Physiol, 1999, 277(1 Pt 1): C132-138.
  12. 12. Egusa H, Iida K, Kobayashi M, et al. Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells. Tissue Eng, 2007, 13(10): 2589-2600.
  13. 13. Timmer NM, van Horssen J, Otte-Holler I, et al. Amyloid beta induces cellular relocalization and production of agrin and glypican-1. Brain Res, 2009, 1260: 38-46.
  14. 14. Oreffo RO, Cooper C, Mason C, et al. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev, 2005, 1(2): 169-178.
  15. 15. Kimelman N, Pelled G, Gazit Z, et al. Applications of gene therapy and adult stem cells in bone bioengineering. Regen Med, 2006, 1(4): 549-561.
  16. 16. Gugala Z, Olmsted-Davis EA, Gannon FH, et al. Osteo-induction by ex vivo adenovirus-mediated BMP-2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.
  17. 17. Gugala Z, Davis AR, Fouletier-Dilling CM, et al. Adenovirus BMP-2 induced osteogenesis in combination with collagen carriers. Biomaterials, 2007, 28(30): 4469-4479.
  18. 18. Sipos W, Pietschmann P, Rauner M. Strategies for novel therapeutic approaches targeting cytokines and signaling pathways of osteoclasto- and osteoblastogenesis in the fight against immune-mediated bone and joint diseases. Curr Med Chem, 2008, 15(2): 127-136.
  19. 19. Alcaraz MJ, Megias J, Garcia-Arnandis I, et al. New molecular targets for the treatment of osteoarthritis. Biochem Pharmacol, 2010, 80(1): 13-21.
  20. 20. Fulci V, Scappucci G, Sebastiani GD, et al. miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol, 2010, 71(2): 206-211.
  21. 21. Luo X, Tsai LM, Shen N, et al. Evidence for microRNA-mediated regulation in rheumatic diseases. Ann Rheum Dis, 2010, 69 Suppl 1: i30-36.
  22. 22. Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.
  23. 23. Babkina IV, Osipov DA, Solovyov YN, et al. Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary osteosarcomas. Bull Exp Biol Med, 2009, 148(2): 246-249.