BMP9介导的骨形成及其机制研究
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摘要
骨再生在众多骨骼系统相关疾病的治疗如骨折愈合、脊柱融合等中起着重要的作用。在促进骨形成的众多调节因子中,骨形成蛋白BMP家族目前已成为研究的重点。BMP属于TGF超家族的一员,在胚胎发育及骨骼形成中扮演着重要的角色,目前人类至少有15种BMP被发现并证实。虽然如今有数种BMP被证实有促进骨形成的作用并应用于临床(主要为BMP2及BMP7),但并未有报道确认其为具有最强成骨作用的BMP。同时由于并非所有重组BMP蛋白都具有生物学活性或都能获得的原因,系统分析所有BMP成骨作用的研究一直未能进行。本研究采用重组腺病毒介导的方式分析14种BMP的成骨作用,发现以小鼠干细胞为载体,感染不同BMP后注射入小鼠大腿中,除传统BMP2及BMP7外, BMP9甚至具有更强的异位成骨的作用。进一步的兔长骨缺损修复实验中,再次证实了BMP9强大的促进骨形成的作用。同时,我们发现BMP3作为一种负调节因子明显抑制BMP2、BMP7介导的骨形成,但对BMP9介导的骨形成并无类似作用。因此我们推测BMP9介导的骨形成并不遵循传统经典BMP信号传导途径而另有它途,并通过基因芯片、Real-time PCR、Western-Blot等方式得到进一步证实。我们的研究为深入探索BMP成骨的作用及机制打下了更坚实的基础,BMP9具有较传统应用的BMP2、7更强大的成骨作用这一新发现为采用基因治疗骨骼系统相关疾病打开了又一扇希望的大门。
Bone regeneration is critical to the effective management of many bone and musculoskeletal disorders, such as fracture healing and spinal fusion. Within series of stimulating factors, the more attention has been focused on the BMPs family. BMPs belong to the TGFb superfamily, and play an important role in embryonic development and bone formation. At least 15 types of BMPs have been identified in humans. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-9 effectively induced orthotopic ossification when AdBMP-transduced osteoblast progenitors were injected into the quadriceps of athymic mice. We confirmed it on a further study by a rabbit bone fracture modle. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2 and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently , and it has been confirmed by gene chip, real time PCR and western blot. Our study expanded the research field of BMP signaling pathway , further more, the use of BMP9 would open a new door for bone regeneration in musculoskeletal disorders.
Bone regeneration is critical to the effective management of many bone and musculoskeletal disorders, such as fracture healing and spinal fusion. Within series of stimulating factors, the more attention has been focused on the BMPs family. BMPs belong to the TGFb superfamily, and play an important role in embryonic development and bone formation. At least 15 types of BMPs have been identified in humans. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-9 effectively induced orthotopic ossification when AdBMP-transduced osteoblast progenitors were injected into the quadriceps of athymic mice. We confirmed it on a further study by a rabbit bone fracture modle. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2 and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently , and it has been confirmed by gene chip, real time PCR and western blot. Our study expanded the research field of BMP signaling pathway , further more, the use of BMP9 would open a new door for bone regeneration in musculoskeletal disorders.
引文
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[2] Anderson HH, Hodges PT, Aguilera XM, Missana L, Moylan PE. 2000. Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage. J Histochem Cytochem 48: 1493-1502
[3] Visser R, Arrabal PM, Becerra J, Rinas U, Cifuentes M. The effect of an rhBMP-2 absorbable collagen sponge-targeted system on bone formation in vivo. Biomaterials. 2009 Apr ; 30 (11) : 2032-7
[4] Desmyter S, Goubau Y, Benahmed N, de Wever A, Verdonk R. The role of bone morphogenetic protein-7 (Osteogenic Protein-1) in the treatment of tibial fracture non-unions. An overview of the use in Belgium. Acta Orthop Belg. 2008 Aug;74(4):534-7
[5] Cheng HW, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, Luu HH, An N, Breyer B,Vanichakarn B, Szatkowski JP, Park JY, and He TC: Osteogenic Activity of the 14 Types of Human Bone Morphogenetic Proteins (BMPs). Journal of Bone and Joint Surgery.2003, 85 (8): 1544-1552
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[11] Reddi G,Gallea AH,Davison AF,et a1. SARA ,a FYVE domain protein that recruits Smad2 to the TGFbeta receptor[J].Cell,2oo6,95(2:779-791
[12] Luca DF ,Barnes KM ,Uyeda JA ,et a1.Regulation of growth plate chondmgcncs is by bone morphogenetic protein-2.Endocrinology。2006,142(1):430-436
[13] Kang Q,Sun MH,Cheng H,et a1.Characterization of the distinct orthotopic bone—form ing activity of 14 BMPs using recombinant adenovirus-mediated gene delivery [J].Gene Ther,2004,11(17):1312-1316
[14] Tylzanowski P,Verschueren K,Huylebroeck D,et a1.Smad-interacting protein is a repressor of liver/bone/kidney alkaline phosphatase transcription in bone morphogenetic protein-induced osteogenic diferentiation of C2C12 cells [J].J Biol Chem,2001,276(43):40001-40007
[15] Groeneveld EH,Burrger EH.Bone morphogenetic proteins in human bone regeneration [J].Eur J Endocrinol,2000,142(1):42-46
[16] Zhang H,Bradley A.Mice deficient for BMP2 am nonviable and have defect in amnion/chorion and cardiac development[J].Development,1996. 122(10):2977-2986
[17] Duprez D,Beu EJ,Richardson MK,et a1.Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb[J].Mech Dev,1996,57(2):145-157
[18] Robert LB,Michael WW,Paul M,et a1.Distal femoral allograft for massive proximal femoral deficiency[J].Acta Orthop Scand,2008,7(1):22-24
[19] He, T.C., Zhou, S., da Costa, L.T., Yu, J., Kinzler, K.W., and Vogelstein, B. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci.U.S.A. 1998. 95: 2509-2514
[20] Romano G. The controversial role of adenoviral-derived vectors in gene therapy programs: where do we stand? Drug News Perspect. 2006 Mar;19(2):99-106
[21] Osten P, Grinevich V, Cetin A. Viral vectors: a wide range of choices and high levels of service. Handb Exp Pharmacol. 2007;(178):177-202
[22] Cheng SL,Lou J,Wright NM ,et a1.In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene. Calcif Tissue Int.2001,68(2):87-94
[23] Visser R, Arrabal PM, Becerra J, Rinas U, Cifuentes M. The effect of an rhBMP-2 absorbable collagen sponge-targeted system on bone formation in vivo. Biomaterials. 2009 Apr ; 30 (11) : 2032-7
[24] Zhang H, Sucato DJ, Welch RD. Recombinant human bone morphogenic protein-2-enhanced anterior spine fusion without bone encroachment into the spinal canal: a histomorphometric study in a thoracoscopically instrumented porcine model. Spine. 2005 Mar 1;30(5):512-8
[25] Desmyter S, Goubau Y, Benahmed N, de Wever A, Verdonk R. The role of bone morphogenetic protein-7 (Osteogenic Protein-1) in the treatment of tibial fracture non-unions. An overview of the use in Belgium. Acta Orthop Belg. 2008 Aug;74(4):534-7
[26] Calori GM, Tagliabue L, Gala L, d'Imporzano M, Peretti G, Albisetti W. Application of rhBMP-7 and platelet-rich plasma in the treatment of long bone non-unions: a prospective randomised clinical study on 120 patients. Injury. 2008 Dec;39(12):1391-402
[27] Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol. 1994 Dec;127(6 Pt 1):1755-1766
[28] Furlan JC, Perrin RG, Govender PV, Petrenko Y, Massicotte EM, Rampersaud YR, Lewis S, Fehlings MG.. Use of osteogenic protein-1 in patients at high risk forspinal pseudarthrosis: a prospective cohort study assessing safety, health-related quality of life, and radiographic fusion. Invited submission from the Joint Section on Disorders of the Spine and Peripheral Nerves, March 2007. J Neurosurg Spine. 2007 Nov;7(5):486-95
[29] Yamaguchi A,Komori T,Suda T.Regulation of osteoblast differentiation mediated by bone morphogenetic proteins,Hedgehogs and Cbfal[J].EndocrRev,2000,21(4):393-411
[30] Massague J, Attisano L, Wrana JL. The TGF-beta family and its composite receptors. Trends Cell Biol. 1994 May;4(5):172-8
[31] Matthews DA, Russell WC. Adenovirus protein-protein interactions: molecular parameters governing the binding of protein VI to hexon and the activation of the adenovirus 23K protease. J Gen Virol. 1995 Aug; 76 ( Pt 8): 1959-69
[32] Suzuki A, Thies RS, Yamaji N, et al. A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10255-9
[33] Imamura T, Maeda S, Hayashi M. [Regulation of BMP signaling]. Clin Calcium. 2006 May ;16 (5):738-44
[34] Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Horm Metab Res. 2005 Jun;37(6):375-9
[35] Rosen V,Cox K,Hattersley G.Bone Morphogenetic Proteins.In Belizikian J P ,Raisz J G,Redan GA(eds).Principles of Bone Biology[M].New York:Academic Press,2006:661 71
[36] Dijke P. Bone morphogenetic protein signal transduction in bone. Curr Med Res Opin. 2006;22 Suppl 1:S7-11
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