• 1 南方醫(yī)科大學南方醫(yī)院創(chuàng)傷骨科（廣州，501515）;;
  • 2 中國醫(yī)學科學院腫瘤醫(yī)院骨科;

目的 組織工程骨的神經(jīng)化能有效促進支架材料內(nèi)血管生成，修復骨缺損。研究降鈣素基因相關(guān)肽（calcitonin gene-related peptide，CGRP）對人臍靜脈血管內(nèi)皮細胞（human umbilical vein endothelial cells，HUVECs）增殖與遷移的作用，進一步揭示組織工程骨的神經(jīng)化促血管生成機制。 方法 體外分離獲取HUVECs，并通過血管性血友病因子（von Willebrand factor，vWF）與CD31 抗原鑒定，取第1 代細胞用于實驗。實驗分為6 組，分別以0（A 組）、1 × 10—12（B 組）、1 × 10—11（C 組）、1 × 10—10（D 組）、1 × 10—9（E 組）、1 × 10—8 mol/L（F 組）濃度CGRP 干預HUVECs。采用細胞免疫熒光觀察HUVECs 的CGRP1 受體（CGRP1 receptor，CGRP1R）表達情況，AlarmarBlue 法動態(tài)檢測各組HUVECs 增殖率，Transwell 小室檢測各組HUVECs 的遷移能力，ELISA 法檢測HUVECs 分泌VEGF 的水平，Westernblot 法檢測其局部黏著斑激酶（focal adhesion kinase，F(xiàn)AK）的表達。 結(jié)果 分離的細胞通過形態(tài)學及vWF、CD31 免疫熒光鑒定為HUVECs，并可見CGRP1R 在細胞質(zhì)和細胞膜表達。CGRP 呈時間- 濃度依賴性刺激HUVECs 增殖；B ～ F組各時間點細胞增殖能力均高于A 組（P  lt; 0.05），F(xiàn) 組各時間點細胞增殖能力最高。B ～ F 組遷移細胞數(shù)均顯著高于A組（P  lt; 0.05），最大增幅達3 倍以上。B ～ F 組VEGF 分泌量均顯著高于A 組（P  lt; 0.05）；C、D 組促進細胞分泌VEGF 的能力最強。Western blot 檢測示，與A組相比，B～F組CGRP刺激HUVECs 3、7、10 d 后，F(xiàn)AK表達明顯增加（P  lt; 0.05）。 結(jié)論 CGRP 對HUVECs 的增殖和遷移有直接促進作用，可能作用機制為CGRP 能促進VEGF 分泌和增加FAK 的表達。

引用本文： 陀泳華 ,郭小磊,張鑫鑫,王釗,周健,張永濤,夏立恒,文軍,金丹. 降鈣素基因相關(guān)肽對人臍靜脈血管內(nèi)皮細胞增殖和遷移的影響及機制研究. 中國修復重建外科雜志, 2012, 26(4): 495-500. doi: 復制

1. Deleu J, Trueta J. Vascularisation of bone grafts in the anterior chamber of the eye. J Bone Joint Surg (Br), 1965, 47: 319-329.
2. Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton. J Bone Miner Res, 2006, 21(2): 183-192.
3. Towler DA. The osteogenic-angiogenic interface: novel insights into the biology of bone formation and fracture repair. Curr Osteoporos Rep, 2008, 6(2): 67-71.
4. Hauge EM, Qvesel D, Eriksen EF, et al. Cancellous bone remodeling occurs in specialized compartments lined by cells expressing osteoblastic markers. J Bone Miner Res, 2001, 16(9): 1575-1582.
5. Hill EL, Elde R. Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res, 1991, 264(3): 469-480.
6. Hukkanen M, Konttinen YT, Santavirta S, et al. Rapid proliferation of calcitonin gene-related Peptide immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling. Neuroscience, 1993, 54(4): 969-979.
7. Aoki M, Tamai K, Saotome K. Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects. Int Orthop, 1994, 18(5): 317-324.
8. 張元平, 崔繼秀, 裴國獻, 等. 神經(jīng)化組織工程骨構(gòu)建的初步觀察. 中華創(chuàng)傷骨科雜志, 2005, 7(1): 60-65.
9. 覃俊君, 王簕, 陳思圓, 等. 血管束、感覺神經(jīng)束植入組織工程骨降鈣素基因相關(guān)肽和受體的時空分布. 中華創(chuàng)傷骨科雜志, 2009, 11(8): 742-746.
10. 劉勇, 裴國獻, 江汕, 等. 組織工程骨神經(jīng)化構(gòu)建的組織學研究. 中國矯形外科雜志, 2009, 17(16): 1246-1249.
11. Lamalice L, Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
12. Li J, Kreicbergs A, Bergstrom J, et al. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia. J Orthop Res, 2007, 25(9): 1204-1212.
13. Maayan C, Bar-On E, Foldes AJ, et al. Bone mineral density and metabolism in familial dysautonomia. Osteoporos Int, 2002, 13(5): 429-433.
14. Akopian A, Demulder A, Ouriaghli F, et al. Effects of CGRP on human osteoclast-like cell formation: A possible connection with the bone loss in neurological disorders? Peptides, 2000, 21(4): 559-564.
15. Bjurholm A, Kreicbergs A, Schultzberg M, et al. Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells. J Bone Miner Res, 1992, 7(9): 1011-1019.
16. Kawase T, Howard GA, Roos BA, et al. Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentrations in UMR 106 osteoblastic cells. Bone, 1995, 16(4 Suppl): 379S-384S.
17. Bernard GW, Shih C. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides, 1990, 11(4): 625-632.
18. Shih C, Bernard GW. Calcitonin gene related peptide enhances bone colony development in vitro. Clin Orthop Relat Res, 1997, (334): 335-344.
19. Collin-Osdoby P. Role of vascular endothelial cells in bone biology. J Cell Biochem, 1994, 55(3): 304-309.
20. Schmid J, Wallkamm B, Hammerle CH, et al. The significance of angiogenesis in guided bone regeneration. A case report of a rabbit experiment. Clin Oral Implants Res, 1997, 8(3): 244-248.
21. Liping Wang, Xiaoyou Shi, Rong Zhao, et al. Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-kB activation, osteocalstogenesis and bone resorption. Bone, 2009, 11(29): 1369-1379.
22. Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
23. Detmar M. Molecular regulation of angiogenesis in the skin. J Inves Dermatol, 1996, 106(2): 207-208.
24. Hamadi A, Bouali M, Dontenwill M, et al. Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397. J Cell Sci, 2005, 118(Pt 19): 4415-4425.
25. Hamid R, Rotshteyn Y, Radadi L, et al. Comparison of alamar blue and MTT asssys for high through-put screening. Toxicol In Vitro, 2004, 18(5): 703-710.
26. Sieg DJ, Hauck CR, Ilic D, et al. FAK integrates growth factor and integrin signals to promote cell migration. Nat Cell Biol, 2000, 2(5): 249-256.
27. Wang HB, Dembo M, Hanks SK, et al. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A, 2001, 98(20): 11295-11300.
28. Lee OH, Lee DJ, Kim YM, et al. Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G (i) protein-linked phospholipase C pathway. Biochem Biophys Res Commun, 2000, 268(1): 47-53.
29. Qin L, Zhang M. Maspin regulates endothelial cell adhesion and migration through an integrin Signaling pathway. J Biol Chem, 2010, 285(42): 32360-32369.
30. Cary LA, Chang JF, Guan JL. Stimulation of cell migration by over-expression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci, 1996, 109(Pt 7): 1787-1794.
31. Kornberg LJ, Shaw LC, Spoerri PE, et al. Focal adhesion kinase overexpression induces enhanced pathological retinal angiogenesis. Invest Ophthalmol Vis Sci, 2004, 45(12): 4463-4469.32 Nakamura J, Shigematsu S, Yamauchi K, et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem Biophys Res Commun, 2008, 374(4): 699-703.
  1. 1. Deleu J, Trueta J. Vascularisation of bone grafts in the anterior chamber of the eye. J Bone Joint Surg (Br), 1965, 47: 319-329.
  2. 2. Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton. J Bone Miner Res, 2006, 21(2): 183-192.
  3. 3. Towler DA. The osteogenic-angiogenic interface: novel insights into the biology of bone formation and fracture repair. Curr Osteoporos Rep, 2008, 6(2): 67-71.
  4. 4. Hauge EM, Qvesel D, Eriksen EF, et al. Cancellous bone remodeling occurs in specialized compartments lined by cells expressing osteoblastic markers. J Bone Miner Res, 2001, 16(9): 1575-1582.
  5. 5. Hill EL, Elde R. Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res, 1991, 264(3): 469-480.
  6. 6. Hukkanen M, Konttinen YT, Santavirta S, et al. Rapid proliferation of calcitonin gene-related Peptide immunoreactive nerves during healing of rat tibial fracture suggests neural involvement in bone growth and remodelling. Neuroscience, 1993, 54(4): 969-979.
  7. 7. Aoki M, Tamai K, Saotome K. Substance P- and calcitonin gene-related peptide-immunofluorescent nerves in the repair of experimental bone defects. Int Orthop, 1994, 18(5): 317-324.
  8. 8. 張元平, 崔繼秀, 裴國獻, 等. 神經(jīng)化組織工程骨構(gòu)建的初步觀察. 中華創(chuàng)傷骨科雜志, 2005, 7(1): 60-65.
  9. 9. 覃俊君, 王簕, 陳思圓, 等. 血管束、感覺神經(jīng)束植入組織工程骨降鈣素基因相關(guān)肽和受體的時空分布. 中華創(chuàng)傷骨科雜志, 2009, 11(8): 742-746.
  10. 10. 劉勇, 裴國獻, 江汕, 等. 組織工程骨神經(jīng)化構(gòu)建的組織學研究. 中國矯形外科雜志, 2009, 17(16): 1246-1249.
  11. 11. Lamalice L, Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
  12. 12. Li J, Kreicbergs A, Bergstrom J, et al. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: A study in rat angulated tibia. J Orthop Res, 2007, 25(9): 1204-1212.
  13. 13. Maayan C, Bar-On E, Foldes AJ, et al. Bone mineral density and metabolism in familial dysautonomia. Osteoporos Int, 2002, 13(5): 429-433.
  14. 14. Akopian A, Demulder A, Ouriaghli F, et al. Effects of CGRP on human osteoclast-like cell formation: A possible connection with the bone loss in neurological disorders? Peptides, 2000, 21(4): 559-564.
  15. 15. Bjurholm A, Kreicbergs A, Schultzberg M, et al. Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells. J Bone Miner Res, 1992, 7(9): 1011-1019.
  16. 16. Kawase T, Howard GA, Roos BA, et al. Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentrations in UMR 106 osteoblastic cells. Bone, 1995, 16(4 Suppl): 379S-384S.
  17. 17. Bernard GW, Shih C. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides, 1990, 11(4): 625-632.
  18. 18. Shih C, Bernard GW. Calcitonin gene related peptide enhances bone colony development in vitro. Clin Orthop Relat Res, 1997, (334): 335-344.
  19. 19. Collin-Osdoby P. Role of vascular endothelial cells in bone biology. J Cell Biochem, 1994, 55(3): 304-309.
  20. 20. Schmid J, Wallkamm B, Hammerle CH, et al. The significance of angiogenesis in guided bone regeneration. A case report of a rabbit experiment. Clin Oral Implants Res, 1997, 8(3): 244-248.
  21. 21. Liping Wang, Xiaoyou Shi, Rong Zhao, et al. Calcitonin-gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-kB activation, osteocalstogenesis and bone resorption. Bone, 2009, 11(29): 1369-1379.
  22. 22. Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res, 2007, 100(6): 782-794.
  23. 23. Detmar M. Molecular regulation of angiogenesis in the skin. J Inves Dermatol, 1996, 106(2): 207-208.
  24. 24. Hamadi A, Bouali M, Dontenwill M, et al. Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397. J Cell Sci, 2005, 118(Pt 19): 4415-4425.
  25. 25. Hamid R, Rotshteyn Y, Radadi L, et al. Comparison of alamar blue and MTT asssys for high through-put screening. Toxicol In Vitro, 2004, 18(5): 703-710.
  26. 26. Sieg DJ, Hauck CR, Ilic D, et al. FAK integrates growth factor and integrin signals to promote cell migration. Nat Cell Biol, 2000, 2(5): 249-256.
  27. 27. Wang HB, Dembo M, Hanks SK, et al. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A, 2001, 98(20): 11295-11300.
  28. 28. Lee OH, Lee DJ, Kim YM, et al. Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G (i) protein-linked phospholipase C pathway. Biochem Biophys Res Commun, 2000, 268(1): 47-53.
  29. 29. Qin L, Zhang M. Maspin regulates endothelial cell adhesion and migration through an integrin Signaling pathway. J Biol Chem, 2010, 285(42): 32360-32369.
  30. 30. Cary LA, Chang JF, Guan JL. Stimulation of cell migration by over-expression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci, 1996, 109(Pt 7): 1787-1794.
  31. 31. Kornberg LJ, Shaw LC, Spoerri PE, et al. Focal adhesion kinase overexpression induces enhanced pathological retinal angiogenesis. Invest Ophthalmol Vis Sci, 2004, 45(12): 4463-4469.32 Nakamura J, Shigematsu S, Yamauchi K, et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem Biophys Res Commun, 2008, 374(4): 699-703.