Vai ai contenuti. | Spostati sulla navigazione | Spostati sulla ricerca | Vai al menu | Contatti | Accessibilità

| Crea un account

Boran, Chiara (2008) Produzione,caratterizzazione e studio dell'attività biologica di TAT-OP1 fattore osteogenetico per l'ingegneria tissutale. [Tesi di dottorato]

Full text disponibile come:

[img]
Anteprima
Documento PDF
2462Kb

Abstract (inglese)

Osteogenic protein-1 (OP-1 or BMP-7) is a member of Bone Morfogenic Proteins’s family (BMPs) and consists of 431 amino acid. BMPs are multi-functional growth factors belonging to the transforming growth factor ? (TGF-?) superfamily. Implicated in a variety of functions as the formation of cartilage and bone, and the development of non-osteogenic tissues (heart, nerve), BMPs are secreted as a precursor approximately four times longer than the mature form, and share a C-terminal distinctive pattern ..C…CXGXC…CC…CXCX.. containing seven cysteines, the active region of the proteins.
In this study we prepared a recombinant fusion protein, called TAT-OP1, including TAT sequence, an Arg rich peptide derived from HIV protein usually used to perform the cell transfection, and a portion of OP-1 sequence. The construct TAT-OP1, 162 aminoacids long, starts with an N-terminal 6His-tag followed by TAT sequence (all together 30 AA), a peptidase specific cleavage site (spanning 6 AA) and the C-terminal OP-1 domain (126 AA) containing the cysteines motive.
Obtained by recombinant DNA technology, the protein TAT-OP1 has been purified by immobilized metal ion affinity chromatography (IMAC) and RP-HPLC, then characterized by SDS-PAGE, aminoacid analysis, UV, CD, and mass spectrometry.
In order to demonstrate the osteogenic potential of recombinant fusion protein TAT-OP1, we treated osteoblastic cell line MC3T3-E1 by using different treatment conditions (pulse- 200 nM and in continous- 5.5, 13.5 and 27 nM treatment).
After 7 and 14 days of cellular treatment performed by application of both stimulation methods, the presence of calcium salt deposits, alkaline phosphatase activity and the expression of osteogenic markers (osteopontin, osteocalcin and Cbfa1/Runx2) were detected by colorimetric and immunoflorescence assays.
To investigate the possible applications of this protein in bone tissue engineering, we developed a research model using adherent fibroblastic cells isolated from umbilical cord blood (UCBMSCs) and a three-dimensional (3D) synthetic scaffold, named Puramatrix Hydrogel TM to mimic the native micro-environment. Moreover, PLGA micro-beads were employed to allow a controlled release of TAT-OP1 with the aim to maintain a suitable level of protein for prolonged times, enhancing its stimulation efficacy.
The primary cultures of UCBMSCs were separated by density-gradient method and were phenotypically characterized in 2D system for the expression of CD105, CD90, CD166, nestin, c-kit, CD31, CD34 and CD38 and for their multi-differentiative potential. As detected on MC3T3-E1 cells, TAT-OP1 demostrated to stimulate nodule calcium formation and the expression of alkaline phosphatase when the cells were treated by both of stimulation methods.
After encapsulation into Puramatrix Hydrogel TM, the cellular response to TAT-OP1 stimulation was evaluated by using electron microscopy analysis, to detect the production of bone like ECM. After 27 days of stimulation with TAT-OP1 (200 nM), microfibrils were observed partially aggregated around the cells. Calcification nodules and Hydroxyapatite crystals were detected only in the cultures encapsulated into Puramatrix Hydrogel TM and treated with PLGA microspheres-controlled release system.
Further investigations will define the utility of this technical approach to improve the in vitro study of osteogenic differentiation and the biological activity of TAT-OP1 for clinical application in the field of bone tissue engineering.


Statistiche Download - Aggiungi a RefWorks
Tipo di EPrint:Tesi di dottorato
Relatore:Grandi, Claudio
Dottorato (corsi e scuole):Ciclo 20 > Scuole per il 20simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:30 Giugno 2008
Anno di Pubblicazione:30 Giugno 2008
Parole chiave (italiano / inglese):TAT-OP1, cellule staminali mesenchimali, differenziamento osteogenico
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/09 Farmaceutico tecnologico applicativo
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:1066
Depositato il:02 Dic 2008
Simple Metadata
Full Metadata
EndNote Format

Bibliografia

I riferimenti della bibliografia possono essere cercati con Cerca la citazione di AIRE, copiando il titolo dell'articolo (o del libro) e la rivista (se presente) nei campi appositi di "Cerca la Citazione di AIRE".
Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

Antonawich F.J., Federoff H.J., Davis J.N. (1999): BCL-2 transduction, using a herpes simplex virus amplicon, protects hippocampal neurons from transient global ischemia. Exp Neurol . 156: 130–137. Cerca con Google

Andrade M.A., Chacon P., Merelo J.J. and Moran F. (1993): Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Engineering. 6: 383-390. Cerca con Google

Babensee J. E., McIntire L. V., Mikos A. G. (2000): Growth factor delivery for tissue engineering. Pharm. Res. 17: 497-504. Baldwin S. P., Saltzman W. M. (1998): Materials for protein deliveryin tissue engineering. Adv. Drug Del. Rev. 33: 71-86. Cerca con Google

Beker-Hapak M., McAllister S., Dowdy S. (2001): TAT-mediated protein transduction domain into mammalian cells. Methods. 24: 247-256. Cerca con Google

Brunner A.M., Gentry L.E., Cooper J.A., Purchio A.F. (1998): Recombinant type 1 transforming growth factor beta precursor produced in Chinese hamster ovary cells is glycosylated and phosphorylated. Mol Cell Biol. 8(5): 2229-2232. Cerca con Google

Celeste A.J., Iannazzi J.A., Taylor R.C., Hewick R.M., Rosen V., Wang E.A., Wozney J.M. (1990): Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci. 87(24): 9843-9847. Check E. (2002): Gene therapy: shining hopes dented - but not dashed. Nature. 420: 735. Cerca con Google

Chen D., Zhao M., Mundy G. (2004): Bone morphogenetic protein. Growth factors. 22(4): 233-241. Cerca con Google

DeLisser H.M., Christofidou-Solomidou M., Strieter R.M., Burdick M.D., Robinson C.S., Wexler R.S., Kerr J.S., Garlanda C., Merwin J.R., Madri J.A., Albelda S.M. (1997): Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am J Pathol. 151(3): 671-677. Dietz G.P., Bahr M. (2004): Delivery of bioactive molecules into the cell: the Trojan horse Approach. Mol Cell Neurosci. 27: 85–131. Cerca con Google

Dolgilevich S., Zaidi N., Song J., Abe E., Moonga B.S., Sun L. (2002): Transduction of TAT fusion proteins into osteoclasts and osteoblasts. Biochem Biophys Res Commun. 299(3): 505-509. Dominici M., Le Blanc K., Mueller I., Slaper-Cortenbach I., Marini F., Krause D., Deans R., Keating A., Cerca con Google

Prockop Dj, Horwitz E. (2006): Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8(4): 315-7. Cerca con Google

Edelhoch H. (1967): Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 6(7): 1948-1954. Cerca con Google

Fackler M.J., Krause D.S., Smith O.M., Civin C.I., May W.S. (1995): Full-length but not truncated CD34 inhibits hematopoietic cell differentiation of M1 cells. Blood. 85(11): 3040-3047. Cerca con Google

Fawell S., Seery J., Daikh Y., Moore C., Chen L.L., Pepinsky B. Barsoum (1994): Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci. 91: 664–668. Cerca con Google

Frankel A.D. and Pabo C.O. (1988): Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 55: 1189-1193. Cerca con Google

Gautschi OP, Frey SP, Zellweger R. (2007): Bone morphogenetic proteins in clinical applications. ANZ J Surg. 77(8): 626-31. Cerca con Google

Green M. and Loewenstein P.M. (1988): Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell. 55: 1179-1188. Cerca con Google

Griffith D. L., Keck P. C., Sampath T. K., Rueger D. C. and Carlson W. D. (1996): Three-dimensional structure of recombinant human osteogenic protein 1: Structural paradigm for the transforming growth factor β superfamily. Proc. Natl. Acad. Sci. 93: 878-883. Cerca con Google

Groppe J., Greenwald J., Wiater E., Rodrizuez-Leon J., Economides A.N., Kwiatkowshi W., Affolter M., Vale W., Belmonte J.C.I. and Choe S. (2002): Structural basis of BMP signaling inhibition by the cystine knot protein Noggin. Nature. 420: 636–642. Cerca con Google

Harwood PJ, Giannoudis PV (2005): Application of bone morphogenetic proteins in orthopaedic practice: their efficacy and side effects. Expert Opin. Drug Saf. 4: 75–89. Hochuli E. (1990): Purification of recombinant proteins with metal chelate adsorbent. Protein. Eng. 12: 87-98. Cerca con Google

Hwang N.S., Kim M.S., Sampattavanich S., Baek J.H., Zhang Z., Elisseeff J. (2006): Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells. Stem Cells. 24(2): 284-291. Cerca con Google

Imamura T., Takase M., Nishihara A., Oeda E., Hanai J., Kawabata, M. and Miyazono K. (1997): Smad6 inhibits signalling by the TGFbeta superfamily. Nature. 389: 622–626. Cerca con Google

Israel DI, Nove J, Kerns KM, Moutsatsos IK, Kaufman RJ. (1992): Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells. Growth Factors. 7(2): 139-150. Cerca con Google

Israel D.I., Nove J., Kerns K.M., Kaufman R.J., Rosen V., Cox K.A., Wozney J.M. (1996): Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors. 13(3-4): 291–300. Cerca con Google

Jainswal N., Haynesworth S.E., Caplan A.I., Bruder S.P. (1997): Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J. Cell. Biochem. 64(2): 295-312. Cerca con Google

Jena N., Martin-Seisdedos C., McCue P., Croce C.M. (1997): BMP7 null mutation in mice: Developmental defects in skeleton, kidney, and eye. Exp Cell Res. 230(1): 28–37. Cerca con Google

Joliot, A., Pernelle C., Deagostini-Bazin H., and Prochiantz A. (1991): Antennapedia homeobox peptide regulates neural morphogenesis. Proc. Natl. Acad. Sci. 88: 1864-1868. Cerca con Google

Kingsley D.M., (1994): The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8(2): 133-146. Cerca con Google

Kim D., Jeon C., Kim J.H., Kim M.S., Yoon C.H., Choi I.S., Kim S.H., Bae Y.S. (2006): Cytoplasmic transduction peptide (CTP): new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo. Exp Cell Res. 312: 1277–88. Cerca con Google

Koenig B.B., Cook J.S., Wolsing D.H., Ting J., Tiesman J.P., Correa P.E., Olson C.A., Pecquetl F., Ventura F., Grant R.A., Chen G., Wrana J., Massague´ J. and Rosenbaum J.S. (1994): Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells. Mol. Cell. Biol. 14: 5961–5974. Cerca con Google

Kordower J.H., Emborg M.E., Bloch J., Ma S.Y., Chu Y., Leventhal L., McBride J., Chen E.Y., Palfi S., Roitberg B.Z., Brown W.D., Holden J.E., Pyzalski R., Taylor M.D., Carvey P., Ling Z., Trono D., Hantraye P., Déglon N., Aebischer P. (2000): Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson′s disease. Science. 290: 767–773. Cerca con Google

Kugler S., Kilic E., Bahr M. (2003): Human synapsin 1 gene promoter confers highly neuron specific longterm transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10: 337–347. Cerca con Google

Laemmli U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Cerca con Google

Langer R., Vacanti J.P. (1993): Tissue engineering. Science. 260: 920-926. Lindsay M.A. (2002): Peptide-mediated cell delivery: application in protein target Validation. Curr Opin Pharmacol. 2: 587–594. Cerca con Google

Macri L., Silverstein D., Clark R.A. (2007): Growth factor binding to the pericellular matrix and its importance in tissue engineering. Adv Drug Deliv Rev. 59(13): 1366-81. Review. Cerca con Google

Mann D.A., Frankel A.D. (1991): Endocytosis and targeting of exogenous HIV-1 Tat Protein. EMBO J. 10: 1733–1739. Cerca con Google

Marcucci G., Baldus C.D., Ruppert A.S., Radmacher M.D., Mrózek K., Whitman S.P., Kolitz J.E., Edwards C.G., Vardiman J.W., Powell B.L., Baer M.R., Moore J.O., Perrotti D., Caligiuri M.A., Carroll A.J., Larson R.A., de la Chapelle A., Bloomfield C.D. (2005): Over expression of the ETS-related gene, ERG, predicts a worse outcome inacute myeloid leukemia with normal karyotype: a Cancer and Leukemia Group B study. J Clin Oncol. 23(36): 9234–9242. Cerca con Google

Moustakas A. and Heldi C. H. (2002): From mono- to oligo-Smads: the heart of the matter in TGFb signal transduction. Genes Dev. 16: 67–1871. Cerca con Google

Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. (1986): Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51 : 263-273. Cerca con Google

Nimni, M. E. (1997): Polypeptide growth factors : targeted delivery systems. Biomaterials. 18: 1201-1225. Cerca con Google

Nishimura R., Kato Y., Chen D., Harris S. E., Mundy G. R. and Yoneda T. (1998): Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J. Biol. Chem. 273: 1872–1879. Cerca con Google

Ozkaynak E., Rueger D.C., Drier E. A., Corbett C., Ridge R. J., Sampath T. K and Oppermann H. (1990): Op-1 cDNA encodes an osteogenic protein in the TGF-β family. Creat. Bio. Mol. 9(7): 2085-2093. Cerca con Google

Porath J., Carlsson J., Olsson I., Belfrage G. (1975): Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258 : 598-599. Cerca con Google

Recchia A., Rota D., Debetto P., Peroni D., Guidolin D., Negro A., Skaper S.D., Giusti P. (2007): Generation of a alpha-synuclein-based rat model of Parkinson's disease. Neurobiol. Dis. 30(1): 8-18. Cerca con Google

Reddi A.H. (2001): Bone morphogenetic proteins: from basic science to clinical applications. J. Bone Joint Surg. Am. 83-A (Suppl. 1): S1-S6. Cerca con Google

Sampath K. T., Maliakal J. C., Hsuschka T., Jones W. K., Sask H., Tucker R. F., White K. H., Coughlin J. E., Tucker M. M., Pang R. H. L., Corbett C., Ozkaynak E., Oppermann H. and Rueger D. C. (1992): Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J. Biol. Cerca con Google

Sato T. (1968): A modified method for lead staining of thin sections. J Electron Microsc. 17(2): 158-159. Cerca con Google

Schwarze S.R., Ho A., Vocero-Akbani A., Dowdy S.F. (1999): In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 285: 1569–1572. Cerca con Google

Semino CE. (2003): Can We Build Artificial Stem Cell Compartments? J Biomed Biotechnol. 2003(3): 164-169. Cerca con Google

Shimazaki K., Urabe M., Monahan J., Ozawa K., Kawai N. (2000): Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemiainduced neuronal death. Gene Ther. 7: 1244–1249. Cerca con Google

Stanchi O., Negro A., Callegaro L. (1997): Expression of osteogenic factor OP-1 in cells of spodoptera frugiperda infected with recombinant baculovirus. United States Patent 5,641,649. Cerca con Google

Studier F.W., Moffatt B.A. (1986): Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-30. Cerca con Google

Suh H. (2000): Tissue restoration, tissue engineering and regenerative medicine. Yonsei Med J. 41(6): 681-4. Review. Cerca con Google

Tabata Y. (2008): Current status of regenerative medical therapy based on drug delivery technology. Reprod Biomed Online. 16(1): 70-80. Review. Cerca con Google

Terada S., Sato M., Sevy A., Vacanti J. P. (2000): Tissue engineering in the twenty-first century. Yonsei Med J. 41(6): 685-91. Review. Cerca con Google

Urist M.R., Iwata H., Ceccotti P.L., Dorfman R. L., Boyd S. D., McDowell R. M. and Chien C. (1973): Bone morphogenesis in implants of insoluble bone gelatin. Proc. Natl. Acad. Sci. 70: 3511–3515. Cerca con Google

Vallejo L.F., Brokelmann M., Marten S., Trappe S., Cabrera-Crespo J., Hoffmann A., Gross G., Weich H.A., Rinas U. (2002): Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. J Biotechnol. 94(2): 185–194. Cerca con Google

Whitaker M. J., Quirk M. A., Howdle S. M. and Shakesheff K. M. (2001): Growth factor release from tissue engineering scaffolds. J. of Pharmacy and Pharmacology. 53: 1427-1437. Cerca con Google

Winnier G., Blessing M., Labosky P.A., Hogan B.L. (1995): Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev.9(17): 2105-2116. Cerca con Google

Woo B.H., Fink B.F., Page R., Schrier J.A., Jo Y.W., Jiang G., DeLuca M., Vasconez H.C., DeLuca P.P. (2001): Enhancement of bone growth by sustained delivery of recombinant human bone morphogenetic protein-2 in a polymeric matrix. Pharm Res. 18(12): 1747-1753. Cerca con Google

Wozney J.M. (2002): Overview of bone morphogenetic proteins. Spine. 27: S2-S8. Cerca con Google

Wright L.R., Rothbard J.B., Wender P.A. (2003): Guanidinium rich peptide transporters and drug delivery. Curr Protein Pept Sci. 4: 105–124. Cerca con Google

Yamashita H., ten Dijke P., Huylebroeck D., Sampath T.K., Andries M., Smith J.S., Heldin C.H. and Miyazono K. (1995): Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J. Cell. Biol. 130: 217–226. Cerca con Google

Yeh L.C., Adamo M.L., Olson M.S., Lee J.C. (1997): Osteogenic protein-1 and insulin-like growth factor I synergistically stimulate rat osteoblastic cell differentiation and proliferation. Endocrinology.138(10): 4181-4190. Cerca con Google

Yoshida Y., Tanaka S., Umemori H., Minowa O., Usui M., Ikematsu N., Hosoda E., Imamura T., Kuno J., Yamashita T., Miyazono K., Noda M., Noda T. and Yamamoto T. (2000): Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell. 103: 1085–1097. Cerca con Google

Zhang H.B., Bradley A. (1996): Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development. 122: 2977–2986. Cerca con Google

Zhang S., Holmes T.C., Lockshin C., Rich A. (1993): Spontaneous assembly of a selfcomplementary oligopeptide to form a stable macroscopic membrane. Proc. Natl. Acad. Sci. 90: 3334- 3338. Cerca con Google

Zhu H., Kavsak P., Abdollah S., Wrana J. and Thomsen G.H.A. (1999): SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature. 400: 687–693. Cerca con Google

Download statistics

Solo per lo Staff dell Archivio: Modifica questo record