成骨生长肽对成骨细胞和骨髓基质细胞增殖分化影响的研究
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摘要
随着口腔种植学的理论不断完善和技术不断发展,使得骨内种植体在临床上得以广泛应用。但是,口腔种植修复也存在着一些亟待解决的问题,例如,如何促进种植体与骨之间形成骨整合,使其能尽快完成种植体上部结构的修复,实现义齿的功能修复等。运用生长因子解决这类问题是目前口腔种植修复学领域研究的热点。成骨生长肽(Osteogenic Growth Peptide,OGP)是近年来发现的一种新的促进有丝分裂的生长因子,已有的研究初步证实了其在成骨和造血等方面具有潜在的应用价值。但是,目前对成骨生长肽的研究尚未涉及其对成骨细胞分化作用机制的研究,同时其对骨髓基质细胞增殖分化方面的研究,国内外也未见报道。
     本课题的研究目的是探讨OGP对成骨细胞和骨髓基质细胞增殖分化功能的影响,初步阐明其促进成骨的作用机制。实验采用成骨细胞和骨髓基质细胞体外培养模型,应用MTT法和流式细胞仪检测不同浓度的OGP对成骨细胞和骨髓基质细胞增殖率和增殖周期的影响。同时应用酶联免疫检测法、免疫细胞化学染色法、激光共聚焦显微镜、透射电子显微镜、半定量逆转录聚合酶链反应(RT-PCR)等方法检测不同浓度的OGP对成骨
Endosteal implant has been widely applied, with the progress of theory and technique of oral implant. But, there have been some pro- blems needing to resolve. For example, how to promote osseointegra- tion between implant and bone around it for completing upper restora- tion to achieve function as soon as possibly? It has been hot region of study in oral implant that utilize growth factors to resolve these pro- blems. People found osteogenic growth peptide as a new mitosis factor can promote bone and blood formation.
    The purpose of our research is to study the effect of osteogenic growth peptide on osteoblast and marrow stromal cells in proliferation and differentiation and elucidate the mechanism of promotion. In our study, we analyzed the effect of different concentration of OGP on the proliferation of osteobalsts and bone marrow stromal cells using MTT and flow cytomitric analysis, utilizing these models of osteoblast and bone marrow stromal cells cultured in vitro. And we studied the influence of different concentration of OGP on the differentiation of osteoblasts and bone marrow stromal cells using
引文
1.张庆诗,刘洪臣。骨生长冈子在种植修复中的应用。中华老年口腔医学杂志,2003,1(2):103—105
    2. Koka S, Vance JB, Maze GL. Bone Growth Factors:Potential for Use as an Osseointegration enhancement technique. Western Society Periodont, 1995, 43(3): 97-104
    3. Giannobile WV. Periodontal tissue engineering by growth factors. Bone, 1996, 19(1): 235-245
    4.毕长华,袁锋杰,邱明才。转化生长因子β与骨。国外医学内分泌学分册,1997,17(1):43—45
    5. Birnbaum RS, Bowsher RR, Wiren KM. Changes in IGF-Ⅰ and-Ⅱ expression and secretion during the proliferation and differentiation of normal rat osteo- blasts. J Endocrinology, 1995, 144:251-259
    6. Horner A, Bord S, Kemp P, et al. Distribution of platelet-derived growth factor (PDGF) a chain mRNA, protein, and PDGF-a receptor in rapidly forming human bone. Bone, 1996, 19 (4): 353-362
    7. Martin L. Growth factors: possible new clinical tools. Acta Orthop Scand, 1996, 67(4): 407-417
    8. Bab I, Gazit D, Chorev M, et al. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity. EMBO J, 1992, 11 (5): 1867-73
    9. Greenberg Z, Chorev M, Muhlrad A, et al. Structural and functional charac- terization of osteogenic growth peptide from human serum: identity with rat and mouse homologs. J Clin Endocrinol Metab, 1995, 80(8): 2330-2335
    10. Greenberg Z, Gavish H, Muhlrad A, et al. Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins. J Cell Biochem, 1997, 65(3): 359-367
    11.司徒镇强,吴军政。细胞培养。世界图书出版公司,2004,3
    12. Masquelier DC, Krummel TA. Morphological characterization of osteoblast-like cell culture isolated from new bone rat calvaria. Calcify Tissue Int, 1990, 47:92-97
    13. Bab 1, Gazit D, Chorev M, et al. H istone H4-related osteogenic growth peptide(OGP): a novel circulating stimulator of osteoblastic activity. EMBO J, 1992, 11:1867-1873
    14.刘鼎新,吕证宝。细胞生物学研究方法与技术。北京医科大学与中国协和医科大学联合出版社,1990
    15. Pittenger MF, Mackay AM, Beck SC, et al. Mutilineage potential of adult human mesenehymal stem cells. Science, 1999, 284(5411): 143-147
    16. Phinney DG, Kopen G, lsaacson RI, et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and diferenciation. J Cell Biochem, 1999, 72:570-585
    17. Dobson KR, Reading L, Haberey M, et al. Centrifugal isolation of bone marow from bone: an improved method for the recovery and quantitation of bone marrow osteogenitor cells from rat tibiae and femurae. Calcif Tissue Int, 1999, 65(5): 411-413
    18. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenehymal progenitor cells. J Cell Physiol, 1999, 181(1): 67-73
    19. Bocci G, Danesi R, Fioravanti A, et al. The effect of osteogenic growth peptide (OGP) on proliferation and adhesion of HEMC-1 human endothelial cells. Pharmacol Res, 2002, 45(1): 21-25.
    20. Chen YC, Bab I, Mansur N, et al. Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)]. J Pept Res, 2000, 56(3): 147-156
    21. Greenberg Z, Chorev M, Muhlrad A, et al. Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge. Biochim Biophys Acta, 1993, 1178(3): 273-80
    22. Sorensen S. Wheat-germ agglutinin method for measuring bone and liver isoenzymes of alkaline phosphatase assessed in postmenopausml osteopo- rosis. Clin Chem, 1988, 34(8): 1636-1640
    23. Genge BR, Sauer GR, Wu LN, et al. Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle mediated mineralization. J Biol Chem, 1988, 263(34): 18513-18519
    24. Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differrentiation interrelationship during progressive development of the osteo- blastic phenotype. 1993, 14(4): 424-441
    25. Chol JY. Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells. J Cell Biochem, 1996, 61(4): 609-618
    26.费琴明,崔大敷,陈统一等。合成成骨生长肽的体内外成骨活性。生物化学与生物物理学报,2001,33(4):415-420
    27. Martin I, Shastri VP, Padera RF, et al. Selective differentiation of mammalian bone marrow stromal cells cultured on three-dimensional polymer foams. J Biomed Mater Res, 2001, 55(2): 229-235
    28. Cheng SL, Yang JW, Rifas L, et al. Differentiation of human bone marrow steogenie stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology, 1994, 134:277-286
    29. Qu Q, Perala-Heape M, Kapanen A, et al. Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. Bone, 1998, 22:201-209
    30. Oxlund H, Mosekilde L, Ouoft G, et al. Reduced concentration of collagen reducible cross links in human trabecullar bone with respect to age and osteoporosis. Bone, 1996, 19:479-484
    31. Garnero P, Sornay-rendu E, Hapuy MC, et al. Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res, 1996, 11:337-349
    32. Petri V, Merjia Pa, Eero V, et al. Expression of matrix genes during incorporation of cancellous bone allografts and autografts. J Clini Orthopaedics and Related Research, 1995, 317:263-272
    33. Cook SD, Salkeld SL, Brinker MR, et al. Use of an osteoinductive biomaterial (rhOP-1) in heal large segmental long bone defects. J Orauma, 1998, 12(6): 407-412
    34. Sun YQ, Ashhurst DE. Osteogenic growth peptide enhances the rate of fracture healing in rabbits. Cell Biol Int, 1998, 22(4): 313-319
    35. Greenberg Z, Chorev M, Muhlrad A, et al. Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge. Biochim Biophys Acta, 1993, 1178(3): 273-280
    36. Lind M, Deleuran B, Thestrup-Pedersen K, et al. Chemotaxis of human osteoblasts effects ofosteotropic growth factors. APMS, 1995, 103(2): 140-146
    37. Cook SD, Salkeld SL, Brinker MR, et al. Use of an osteoinductive biomaterial (rhOP-1) in heal large segmental long bone defects. J Orauma, 1998, 12(6): 407-412
    38. Geng WD, Boskovic G, Fultz ME, et aL Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells. J Cell Physiol 2001; 189:216-228
    39.符民桂,唐朝枢。细胞Ca~(2+)信号对基因转录的调节。国外医学生理、病理科学与临床分册,1999,19(5):348-352
    40. Janmey PA. The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol Rev, 1998, 78:763-781
    41. Maniotis AJ, Chen CS, Ingber DE. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci USA, 1997, 94(3): 849-854
    42. Guan YY, Kwan CY, He H, et al. Effects of Panax notoginseng saponins on receptor-operated Ca~(2+) channel in vascular smooth muscle. Acka Pharrna. Sinica, 1994, 15; 385-392
    43. Charms GT, Horton MA. Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophysical Journal, 2002, 82: 2970-2981
    44. Andrew Grey, Yan Chen, Indu PaliwaL, et al. Evidence for a functional association between phosphatidylinosito13-kinase and c-sre in the spreading response of osteoclasts to colony-stimulating factor-1. Endocrinology, 2000, 141: 2129-2138
    45. Rijikers GT, Justerment LB, Griffioen AW, et al. Improved method for measuring intracellular Ca~(2+) with fluo-3. Cytometry, 1990, 11:923-927
    46.霍霞,鲍永耀,黄辉等。激光扫描共聚焦显微镜和Fluo-3/AM检测细胞内游离钙。激光杂志,1998,19:35—37
    47. Drissi H, Luc Q,Shakoori R, et al. Transcriptional autoregulation of thebone related CBFA1/RUNX2 gene.J Cell Physiol, 2000, 184(3):341-350
    48. Ahdjoudj S, Lasmoles F, Oyajobi BO, et al. Reciprocal control of osteoblast/chondroblast and osteoblast/ adipocyte differentiation of multipotential clonal human marrow stromal F/STRO1 cells. J cell Biochem, 2001, 1(1): 23-38
    49. Fujita T, Izumo N, Fukuyama T, et al. Phosphate provides an extracelllular signal that drives nuclear export of Runx2/Cbfal. Biochem Biophys Res Commun, 2001,280(1): 348-352
    50. 孟昭亨。骨钙素及其临床意义。中华内分泌代谢杂志, 1992, 8: 41-44
    
    51. Qu Q, Perala-Heape M, Kapanen A, et al. Estrogen enhances differentiation of osteoblast in mouse bone marrow culture. Bone, 1998, 22: 201-209
    
    52. Ryoo HM, Wijnen AJ, stein J, et al. Detection of a proliferation specific gene during development of the osteoblast phenotype by mRNA differential display. J cell Biochem, 1997,64: 106-116
    
    53. Collin P, Nefussi JR, Wetterwald A, et al. Expression of collagen, osteo- calcin, and bone alkaline phosphatase in a minesliying rat osteoblastic cell culture. Calcif Tissue Int, 1992,50: 175-183
    
    54. Komori T, Yagi H, Nomura S, et al. Targeted disruption of cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 1997, 89: 755-764
    
    55. Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfal: A transcriptional activator of osteoblast differentiation. Cell, 1997, 89: 747-754
    
    56. Owen M, Friendenstein AJ. Stromal stem cells: Marrow-derived osteogenic precursors. Ciba Found Symp, 1998, 136: 42-60
    
    57. Komeri T, Yagi H, Nomurs S, et al. Targeted disruption of Cbfal results in a com plere lack of bone form ation owing to m aturational arrest of osteolasts. Cell, 1997, 89:755-764
    
    58. Kobayashi K, Krebsbach P, Bianco P, et al. Osteogenic imprinting up-stream of marrow stromal cell differentiation. J Cell Biochem, 2000, 78: 391-403
    
    59. Ducy P, Starbuck M, Priemel M, et al. A Cbfal-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev, 1999, 13(8): 1025-1036
    
    60. Fagenholz PJ, Warren SM, Greenwald JA, et al. Osteoblast gene expression is differentially regulated by TGF beta iscforms. J Craniofac Surg, 2001,12(2): 183-190
    
    61. Gallea S, Lallemand F, Atfi A, et al. Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone, 2001, 28(5): 491-498
    
    62. Chevalley Th, Rizzoli R, Manen D, et al. Arginine increases insulin-like growth factor-1 production and collagen synthesis in osteoblast-like cells. Bone, 1998, 232: 103-109
    
    63. Kobayashi H, Gao YH, Ueta C, et al. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev, 1999, 13(8): 1025-1036
    
    64. Xiao GZ, Jiang D, Thomas P, et al. MAPK pathway activate and phos- phorylate the osteoblast-specific transcription factor, Cbfal. J Biol Chem, 2000, 275(6): 4453-4459
    
    65. Tintut Y, Parhami F, Le V, et al. Inhibition of osteoblast-specific trans- cription factor Cbfal by the cAMP pathway in osteoblastic cells. Ubiquitin/ proteasome-dependent regulation. J Biol Chem, 1999, 274(41): 28875-28879
    
    66. Gao YH, Shinki T, Yuasa T, et al. potential role of Cbfal, an essential transcriptional factor for osteoblast differentiation, in osteoclastogenesis: regulation of mRNA expression of osteoclast differentiation factor(ODF). Biochem Biophys Res Comm, 1998, 252(3): 697-702
    1. Bab I, Gazit D, Chorev M, et al. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity: EMBO J, 1992, 11(5): 1867-73
    2. Greenberg Z, Chorev M, Muhlrad A, et al. Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs. J Clin Endocrinol Metab, 1995, 80(8): 2330-5
    3. Greenberg Z, Chorev M, Muhlrad A, et al. Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge. Biochim Biophys Acta, 1993, 1178(3): 273-80
    4. Bab IA, Einhorn TA. Regulatory role of osteogenic growth polypeptides in bone formation and hemopoiesis. Crit Rev Eukaryot Gene Expr, 1993, 3(1): 31-46
    5. Bab I, Garish H, Namdar-Attar M, et al. Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells. J Pept Res, 1999, 54(5): 408-14
    6. Chen YC, Bah I, Mansur N, et al. Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)]. J Pept Res, 2000, 56(3): 147-56
    7. Bab I, Smith E, Gavish H, et al. Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA. J Biol Chem, 1999, 274(20): 14474-81
    8. Greenberg Z, Gavish H, Muhlrad A, et al. Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins. J Cell Biochem, 1997, 65(3): 359-67
    9. Bab IA. Regulatory role of osteogenic growth peptide in proliferation, osteogenesis, and hemopoiesis. Clin Orthop Relat Res, 1995, 313:64-8
    10. Sun YQ, Ashhurst DE. Osteogenic growth peptide enhances the rate of fracture healing in rabbits. Cell Biol Int, 1998, 22(4): 313-9
    11.费琴明,崔大敷,陈统一,等。合成成骨生长肽的体内外成骨活性。生物化学与生物物理学报,2001,33(4):415-20
    12. Robinson D, Bab I, Nevo Z. Osteogenic growth peptide regulates proliferation and osteogenic maturation of human and rabbit bone marrow stromal cells. J Bone Miner Res, 1995, 10(5): 690-6
    13. Gabarin N, Gavish H, Muhlrad A, et al. Mitogenic G(i) protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: activation by C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)] and attenuation of activation by cAMP. J Cell Biochem, 2001, 81(4): 594-603
    14. Fazzi R, Galimberti S, Testi R, et al. Bone and bone marrow interactions: hematological activity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide. Ⅱ. Action on human hematopoietic stem cells. Leuk Res, 2002, 26(9): 839-48
    15. Gurevitch O, Slavin S, Muhlrad A, et al. Osteogenic growth peptide increases blood and bone marrow cellularity and enhances engraftment of bone marrow transplants in mice. Blood. 1996, 88(12): 4719-24
    16. Fazzi R, Testi R, Trasciatti S, et al. Bone and bone-marrow interactions: haematological actiyity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide. Mobilizing properties on white blood cells and peripheral blood stem cells in mice. Leuk Res, 2002, 26(1): 19-27
    
    17. Fazzi R, Galimberti S, Testi R, et al. Bone and bone marrow interactions: hematological activity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide. II. Action on human hematopoietic stem cells. Leuk Res, 2002, 26(9): 839-48
    
    18. Mattii L, Fazzi R, Moscato S, et al. Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA. J Cell Biochem, 2004, 93(6): 1231-41
    
    19. Bocci G, Danesi R, Fioravanti A, et al. The effect of osteogenic growth peptide (OGP) on proliferation and adhesion of HEMC-1 human endothelial cells. Pharmacol Res, 2002, 45(1): 21-5
    1.路艳蒙,傅文玉,朴英杰,等。人骨髓间充质干细胞的超微结构。电子显微镜学报,2002,21(4):373
    2. Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchyrnal stem cells during extensive subculativation and following cryopreservation. J Cell Biochem, 1997, 64(2): 278
    3. Aubin JE. Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interaction in osteoblast differentiation. Cell Tissue Res, 1999, 72(3): 396
    4. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med, 2001, 226(6):507
    5.张文志,孔荣,方诗元,等。兔自体骨髓间充质干细胞体内复合移植的成骨研究。 临床骨科杂志, 2002, 5(2): 81
    
    6. Zohar R, Sodek I, McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997, 90(9): 3471
    
    7. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411): 143
    
    8. Milne M, Quail JM, Baran DT.Dexamethasone stimulates osteogenic differentiation in vertibral and femoral bone marrow cell culture xomparison of IGF-I gene expression. J cell Biochem, 1998, 71(3): 382
    
    9. David CC, Reiner C, Catla M, et al. Rapid expansion of recycling stems cells in cultures of plastic adherent cells from human bone marrow. Proc Natl Acad Sci USA, 2000, 97(7): 3213
    
    10. 金岩。组织工程学与相关技术。
    
    11. Rickard DJ, Sullivan TA, Shenker BJ, et al. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell culture by dexamethasone and BMP-2.DevBiol, 1994, 161(1): 218
    
    12. Akira Y, Takenobu K, Tohru I, et al. Recombinant human bone morphogenetic protein-2 stimulate osteolbastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol, 1996, 113(2): 681
    
    13. Hanada K, Dennis JE, Caplan AI, et al. Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res, 1997, 35(12): 1606
    
    14. Martin I, Padera RF, Vnnjak-Novakovic G, et al. In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues. J Orthop Res, 1998, 16(2): 181
    
    15. Locklin RM, Williamson MC, Beresfird JN, et al. In vitro effects of growth factors and dexamethesone on rat marrow stromal cells. Clin Orthop, 1995, 313(4): 27
    
    16. Andrades JA, Han B, Becerra J, et al. A reconabinant human TGF-beta1 fusion protein with collagen binding domain promotes migration growth and differentiation of bonemarrow mesenchymal cells. Exp Cell Res, 1999, 250(2): 485
    
    17. Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro by stromal cell obtained from bone marrow of young adult rat. Cell Tissue Res, 1988, 254(3): 317
    
    18. Cheng SL, Zhang SF, Mohan S, et al. Regulation of insulin-like growth factors I and 11 and their binding proteins in human bone marrow stromal cell by dexamethasone. J Cell Biochem, 1998, 71(3): 449