降鈣素基因相關肽對人臍靜脈血管內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》

作者：

陀泳華 ¹ , 郭小磊 ¹ , 張鑫鑫 ² , 王釗 ¹ , 周健 ¹ , 張永濤 ¹ , 夏立恒 ¹ , 文軍 ¹ , 金丹 ¹

1 南方醫科大學南方醫院創傷骨科（廣州，501515）;;
2 中國醫學科學院腫瘤醫院骨科;

關鍵詞：

降鈣素基因相關肽血管生成細胞遷移細胞增殖骨組織工程

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目的組織工程骨的神經化能有效促進支架材料內血管生成，修復骨缺損。研究降鈣素基因相關肽（calcitonin gene-related peptide，CGRP）對人臍靜脈血管內皮細胞（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，FAK）的表達。結果分離的細胞通過形態學及vWF、CD31 免疫熒光鑒定為HUVECs，并可見CGRP1R 在細胞質和細胞膜表達。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 組各時間點細胞增殖能力最高。B ～ F 組遷移細胞數均顯著高于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，FAK表達明顯增加（P lt; 0.05）。結論 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. 降鈣素基因相關肽對人臍靜脈血管內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. 神經化組織工程骨構建的初步觀察. 中華創傷骨科雜志, 2005, 7(1): 60-65.
9.	覃俊君, 王簕, 陳思圓, 等. 血管束、感覺神經束植入組織工程骨降鈣素基因相關肽和受體的時空分布. 中華創傷骨科雜志, 2009, 11(8): 742-746.
10.	劉勇, 裴國獻, 江汕, 等. 組織工程骨神經化構建的組織學研究. 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. 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. 神經化組織工程骨構建的初步觀察. 中華創傷骨科雜志, 2005, 7(1): 60-65.
9. 覃俊君, 王簕, 陳思圓, 等. 血管束、感覺神經束植入組織工程骨降鈣素基因相關肽和受體的時空分布. 中華創傷骨科雜志, 2009, 11(8): 742-746.
10. 劉勇, 裴國獻, 江汕, 等. 組織工程骨神經化構建的組織學研究. 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.

《中國修復重建外科雜志》

降鈣素基因相關肽對人臍靜脈血管內皮細胞增殖和遷移的影響及機制研究

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《中國修復重建外科雜志》

降鈣素基因相關肽對人臍靜脈血管內皮細胞增殖和遷移的影響及機制研究

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