誘導hBMSCs 成骨分化中微小RNA 和靶基因表達水平的變化_《中國修復重建外科雜志》

作者：

魏均強 ¹ , 張伯勛 ¹ , 鄭曉飛 ² , 陳華 ¹ , 田學忠 ¹ , 唐佩福 ¹ , 宋青 ³ , 黎檀實 ³

解放軍總醫院（北京，100853）1 骨科，3 急救部;;
2 軍事醫學科學院輻射醫學研究所;

關鍵詞：

微小RNA 靶基因 hBMSCs 成骨誘導分化

視頻：

導出 下載 收藏 掃碼 引用

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

目的明確萎縮性骨不連組織中表達上調的數種微小RNA（micro RNA，miRNA）與其相應靶基因mRNA、蛋白在hBMSCs 成骨分化過程中的表達變化趨勢和生物學功能。方法取自體髂骨植骨手術患者的髂骨骨髓血，采用密度梯度離心法分離培養hBMSCs。取第4 代hBMSCs 以成骨誘導培養液誘導成骨分化，分別提取0、12 h，1、2、4、7、14 d 時的細胞總RNA 和蛋白，進行miRNA 的實時定量PCR（quantitative real-time PCR，qRT-PCR）、相應靶基因mRNA 的qRT-PCR 和蛋白Western blot 檢測。結果誘導hBMSCs 成骨分化時，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 和蛋白表達在多數時間點同對照（0 h）相比增加（BMP-2 在12 h 和1 d 時下降），1 ～ 7 d 變化最為顯著。不同時間點的miRNA、靶基因mRNA 和蛋白表達水平存在差異，其中hsa-miRNA-149 和hsa-miRNA-654-5p miRNA 含量的變化趨勢與各自靶基因ALPL 和BMP-2 的mRNA 及蛋白表達水平總體上成負相關（P lt; 0.05），hsa-miRNA-221 與其靶基因PDGF-A 的變化趨勢無明顯負相關關系（P gt; 0.05）。結論誘導hBMSCs 成骨分化過程中，hsa-miRNA-149 和hsa-miRNA-654-5p 對其相應靶基因ALPL 和BMP-2 的mRNA 及蛋白存在密切調控關系。

引用本文： 魏均強 ,張伯勛,鄭曉飛,陳華,田學忠,唐佩福,宋青,黎檀實. 誘導hBMSCs 成骨分化中微小RNA 和靶基因表達水平的變化. 中國修復重建外科雜志, 2012, 26(4): 483-487. doi: 復制

1. 魏均強. miRNA在萎縮性骨不連中的調控作用及其分子生物學機制. 北京: 中國人民解放軍軍醫進修學院, 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. 骨髓基質細胞誘導分化成骨方法及相關研究進展. 中國組織工程研究與臨床康復, 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. 魏均強. miRNA在萎縮性骨不連中的調控作用及其分子生物學機制. 北京: 中國人民解放軍軍醫進修學院, 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. 骨髓基質細胞誘導分化成骨方法及相關研究進展. 中國組織工程研究與臨床康復, 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資料與方法

1.1一般資料

1.2方法

1.3察指標和判定標準

1.4統計學方法

2結果

2.1兩組患者療效比較

2.2兩組患者換藥次數、導管留置時間、治愈時間比較

2.3兩組患者治療前后血清PCT、CRP、WBC水平比較

3討論

《中國修復重建外科雜志》

誘導hBMSCs 成骨分化中微小RNA 和靶基因表達水平的變化

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

1.	魏均強. miRNA在萎縮性骨不連中的調控作用及其分子生物學機制. 北京: 中國人民解放軍軍醫進修學院, 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. 骨髓基質細胞誘導分化成骨方法及相關研究進展. 中國組織工程研究與臨床康復, 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.

《中國修復重建外科雜志》

誘導hBMSCs 成骨分化中微小RNA 和靶基因表達水平的變化

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

摘要全文圖表視頻參考文獻施引文獻補充材料