含醣胺素結合區及RGD序列之人工細胞外間質蛋白

簡易檢索 / 詳目顯示

回結果列表

研究生：	余蓉荃 Jung-Chuan Yu
論文名稱：	含醣胺素結合區及RGD序列之人工細胞外間質蛋白 Artificial Extracellular Matrix Proteins Containing Glycosaminoglycan-binding Domain and RGD Motif
指導教授：	吳夙欽 Suh-Chin Wu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	71
中文關鍵詞：	日本腦炎病毒、醣胺素結合區、RGD 、造骨細胞、軟骨細胞、人工細胞外間質
外文關鍵詞：	JEV, Glycosaminoglycan-binding domain, RGD, Osteoblast, Chondrocyte, Artificial Extracellular Matrix
相關次數：	點閱：2 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

在動物細胞中，細胞外有由細胞分泌出去的多種蛋白質，構成細胞外間質（extracellular matrix，ECM），這些蛋白質中含有結構性蛋白質、蛋白醣（proteoglycans）及附著性蛋白（adhesive proteins）。主要的結構性蛋白質有膠原蛋白（collagens）及elastins，與細胞外之蛋白醣結合成網狀結構，結構性蛋白質位於網中與細胞上之附著性蛋白結合，附著性蛋白有fibronectins 、laminins及tenascins。這些細胞外間質會影響與其接處細胞的行為，包括生存、發育、移動、增生、形狀和功能。可藉由設計及表現人工基因的技術來製作可調控細胞行為的人工細胞外間質（artificial ECM）。在這篇研究中，將帶有不同來源的RGD序列之片段與日本腦炎病毒套膜蛋白上的醣胺素結合區合併作為人工細胞外間質，並檢驗這些人工細胞外間質對類造骨細胞（osteoblast-like MG-63 cells）及軟骨細胞（chondrocytes NHAC-kn）的貼附、增生及分化之影響。所設計的三個人工細胞外間質包括：387RGDS是一個修改成RGDS序列的日本腦炎病毒套膜蛋白片段、Tri-FN10是一個將含有兩個日本腦炎病毒套膜蛋白上的醣胺素結合區及fibronectin上含有RGD序列的15個胺基酸合併後重複三次的蛋白質、TNC-FN3是一個混合tenascin-C 上的第十個fibronectin type II repeat 與兩個日本腦炎病毒套膜蛋白上的醣胺素結合區之蛋白質。相對於沒有人工細胞外間質的培養條件下，設計的三個人工細胞外間質均可增加MG-63及NHAC-kn細胞貼附及增生，但是TNC-FN3的效果最佳，其次為Tri-FN10，第三為387RGDS。

The extracellular matrix (ECM) influences the survival, development, migration, proliferation, shape, and function of the cells that contact it. Through the design and expression of artificial genes, it is possible to prepare artificial ECM (aECM) proteins with domains chosen to modulate cellular behaviors. The objective of this study was to examine what combination of the RGD-containing fragments with two JEV E protein heparin-binding domains, served as aECM proteins, were suitable materials for human osteoblast-like MG-63 cells and human articular chondrocytes NHAC-kn attachment, proliferation and differentiation on tissue culture plate, polycaprolactone membrane, Cytodex 1 and Plastic Plus microcarriers. 387RGDS was a RGDS modified JEV E protein fragment, Tri-FN10 was a three-repeat protein of heparin-binding domains and fibronectin RGD-containing peptide, and TNC-FN3 was a fusion protein of the third fibronectin type III repeat of tenascin-C with heparin-binding domains. The three aECM proteins had a trend of: TNC-FN3> Tri-FN10> 387RGDS on attachment and proliferation enhancing effects on MG-63 and NHAC-kn cells. And the osteocalcin, osteopontin, and collagen type I expression of MG-63 cells were maintained on these three aECM proteins coated materials. Chondrocytes cultured on aECM proteins coated and uncoated 3D cytodex 1 surfaces and with TNC-FN3 coated tissue culture plate could re-differentiate to phenotype of type II collagen expression.

Chinese Abstract
English Abstract
Acknowledgement
Content
1. Introduction
1.1. Extracellular Matrix
1.1.1. The General Overview of Extracellular Matrix
1.1.1.1. Fibronectin
1.1.1.2. Tenascin-C
1.1.2. The Influences of Extracellular Matrix on Osteoblasts
1.1.3. The Influences of Extracellular Matrix on Chondrocytes
1.2. Artificial Extracellular Matrix
1.2.1. The Role of Artificial Extracellular Matrix in Biomaterials
1.2.2. RGD Cell-Binding Domains of Artificial Extracellular Matrix
1.2.3. Heparin-Binding Domains of Artificial Extracellular Matrix
1.3. Biodegradable Synthetic Polymers
1.3.1. The Overview of Biodegradable Synthetic Polymers
1.3.2. Polycaprolactone
1.4. Three-Dimensional Cultures in Bone and Cartilage Tissue Engineering
2. Materials and Methods
2.1. Molecular Methods
2.1.1. RNA Extraction and cDNA Synthesis
2.1.2. Polymerase-Chain Reaction
2.2. Artificial Extracellular Matrix Proteins Construction
2.2.1. 387RGDS
2.2.2. Tri-FN10
2.2.3. TNC-FN3
2.3. Artificial Extracellular Matrix Proteins Expression and Purification
2.4. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
2.5. Cell Culture
2.5.1. Static Culture
2.5.2. Microcarrier Culture
2.6. Polycaprolactone Membrane Preparation
2.7. Artificial ECM Proteins Coating
2.8. Attachment Assay
2.9. Proliferation Assay
2.10. Gene Expression Analysis
3. Results and Discussion
3.1. Artificial Extracellular Matrix Proteins Expression and Purification
3.2. The Effects of Artificial ECM Proteins to Osteoblast-like cells on 2D and 3D cultures
3.2.1. The Attachment of Osteoblast-like cells
3.2.2. The Proliferation of Osteoblast-like cells
3.2.3. The Gene Expression of Osteoblast-like cells
3.3. The Effects of aECM Proteins to Chondrocytes on 2D and 3D cultures
3.3.1. The Attachment of Chondrocytes
3.3.2. The Proliferation of Chondrocytes
3.3.3. The Differentiation of Chondrocytes
4. Conclusions
5. Appendix
5.1. Artificial ECM Proteins DNA Sequence
5.1.1. 387RGDS DNA Sequence
5.1.2. Tri-FN10 DNA Sequence
5.1.3. TNC-FN3 DNA Sequence
5.2. Artificial ECM Protein Sequence
5.2.1. 387RGDS Protein Sequence
5.2.2. Tri-FN10 Protein Sequence
5.2.3. TNC-FN3 Protein Sequence
Appendix Table 1. The Comparison of Cytodex 1 and Plastic Plus Microcarriers
Appendix Table 2. Primer List for Constructions
Appendix Table 3. Gene Expression was Detected By PCR
Appendix Figure 1. The Chemical Structure of Polycaprolactone
Appendix Figure 2. 387RGDS Cloning
Appendix Figure 3. Tri-FN10 Cloning
Appendix Figure 4. TNC-FN3 Cloning
References
Table 1. The Time for 50% of Osteoblast-Like MG-63 Cells Attachment
Table 2. The Time For 50% of Chondrocytes NHAC-Kn Attachment
Figure 1. Artificial Extracellular Matrix (aECM) Protein Constructions.
Figure 2. The aECM Proteins Expression
Figure 3. The Purities of aECM Proteins after Purification
Figure 4. The Cell morphology of Attached Osteoblast-Like Cells
Figure 5. The Attachment of Osteoblast-Like Cells on 2D Culture
Figure 6. The Attachment of Osteoblast-Like Cells on 3D Culture
Figure 7. The Proliferation of Osteoblast-Like Cells on 2D Culture
Figure 8. The Proliferation of Osteoblast-Like Cells on 3D Culture
Figure 9. Gene Analysis of Osteoblast-Like Cells
Figure 10 The Morphology of Attached Chondrocytes
Figure 11. The Attachment of Chondrocytes on 2D Culture
Figure 12. The Attachment of Chondrocytes on 3D Culture
Figure 13. The Proliferation of Chondrocytes on 2D Culture
Figure 14. The Proliferation of Chondrocytes on 3D Culture
Figure 15. Gene Analysis of Chondrocytes

                                

Adams CS, Shapiro IM. 2003. Mechanisms by which extracellular matrix components induce osteoblast apoptosis. Connect Tissue Res. 44:230-239.
Adams JC, Watt FM. 1993. Regulation of development and differentiation by the extracellular matrix. Development. 117:1183-1198.
Anselme K. 2000. Osteoblast adhesion on biomaterials. Biomaterials. 21:667-681.
Ashammakhi N, Rokkanen P. 1997. Absorbable polyglycolide devices in trauma and bone surgery. Biomaterials. 18: 3-9.
Aubin JE. 1998. Bone stem cells. J Cell Biochem Suppl. 30-31:73-82.
Behravesh E, Yasko AW, Engle PS, Mikos AG. 1999. Synthetic biodegradable polymers for orthopaedic applications. Clin Orthop. 367S: 118-185.
Blunk T, Sieminski AL, Gooch KJ, Courter DL, Hollander AP, Nahir AM, Langer R, Vunjak-Novakovic G, Freed LE. 2002. Differential effects of growth factors on tissue-engineered cartilage. Tissue Eng. 8:73-84.
Burg KJL, Porter S, Kellam JF. 2000. Biomaterials development for bone tissue engineering. Biomaterials. 21:2347-2359.
Cao T, Ho KH, Teoh SH. 2003. Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling. Tissue Eng. 9:S103-112.
Chiquet-Ehrismann R. 2004. Tenascins. Int J Biochem Cell Biol. 36:986-990.
Chiquet-Ehrismann R, Chiquet M., 2003. Tenascins: regulation and putative functions during pathological stress. J Pathol. 200:488-499.
Chiquet-Ehrismann R, Tucker RP. 2004. Connective tissues: signalling by tenascins.
Int J Biochem Cell Biol. 36:1085-1089.
Clover J, Gowen M. 1994. Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? Bone. 15:585-591.
Craig WS, Cheng S, Mullen DG, Blevitt J, Pierschbacher MD. 1995. Concept and progress in the development of RGD-containing peptide pharmaceuticals. Biopolymers. 37:157-175.
Dalton BA, McFarland CD, Underwood PA, Steele JG. 1995. Role of the heparin binding domain of fibronectin in attachment and spreading of human bone-derived cells. J Cell Sci. 108:2083-2092.
Damsky CH. 1999. Extracellular matrix-integrin interactions in osteoblast function and tissue remodeling. Bone. 25:95-96.
De Arcangelis A, Georges-Labouesss E. 2000. Integrin and ECM functions: roles in vertebrate development. Trends Genet. 16:389-395.
Dee KC, Andersen TT, Bizios R. 1998 Design and function of novel osteoblast-adhesive peptides for chemical modification of biomaterials. J Biomed Mater Res. 40:371-377.
Eyre D. 2002. Collagen of articular cartilage. Arthritis Res. 4:30-35.
Friedl P, Brocker EB. 2000. The biology of cell locomotion within three-dimensional extracellular matrix. Cell Mol Life Sci 57:41-64.
Frondoza C, Sohrabi A, Hungerford D. 1996. Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture. Biomaterials. 17:879-888.
George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H., Hynes RO. 1993. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119:1079-1091.
Grzesik WJ, Robey PG. 1994. Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro. J Bone Miner Res. 9:487-496.
Guntillake PA, Adhikari R. 2003. Biodegradable synthetic polymers for tissue engineering. Eur Cell Mater. 5:1-16
Hayashi T. 1994. Biodegradable polymers for biomedical applications. Prog Polymer Sci. 19:663-702.
Hoffman N. 1993. To do tissue culture in two or three dimensions? That is he question. Stem Cells. 11:105-111
Howard GA, Turner RT, Puzas JE, Nichols F, Baylink DJ. 1983. Bone cells on microcarrier spheres. JAMA. 249:258-259.
Hubbell J. 1995. Biomaterials in tissue engineering. Biotechnology. 13:565-576.
Huebsch JB, Fields GB, Triebes TG, Mooradian DL. 1996. Photoreactive analog of peptide FN-C/H-V from the carboxy-terminal heparin-binding domains of fibronectin supports endothelial cell adhesion and spreading on biomaterial surfaces. J Biomed Mater Res. 31:555-567.
Hutmacher DW, Zein I, Teoh SH, Ng KW, Schantz JT, Leahy JC. 2000. Design and fabrication of a 3D scaffold for tissue engineering bone. In: Agrawal CM, Parr JE, Lin ST, eds. Synthetic Bioabsorbable Polymers for Implants, ASTM STP 1396. West Conshohocken, PA: American Society for Testing and Materials. p. 152–167.
In’tVeld PJA, Velner EM, Van DeWhite P, Hamhuis J, Dijkstra PJ, Feijen J. 1997. Melt block copolymerisation of e-caprolactone and L-Lactide. J. Polym Sci Part 1 Polymer Chem. 35:219-226.
Kao WJ. 1999. Evaluation of protein-modulated macrophage behavior on biomaterials: Designing biomimetic materials for cellular engineering. Biomaterials. 20:2213-2221.
Kjellen L, Lindahl U. 1991. Proteoglycans: structures and interactions. Annu Rev Biochem. 60:443-475.
Knupp C, Squire JM. 2003. Molecular packing in network-forming collagens. ScientificWorld Journal. 23:558-577.
Kohn J, Langer R. 1997. Bioresorbable and bioerodible materials. In: An Introduction to Materials in Medicine. Ratner BD, Hoffman AS, Schoen FJ, Lemon JE, eds. Academic Press, San Diego. p. 65-73.
Kronenthal RL. 1975. Biodegradable polymers in medicine and surgery. Polymer Sci Technol. 8:119-137.
Kosmehl H, Berndt A, Katenkamp D. 1996. Molecular variants of fibronectin and laminin: structure, physiological occurrence and histopathological aspects. Virchows Arch. 429:311-322.
Kresse H, Schonherr E. 2001. Proteoglycans of the extracellular matrix and growth control. J Cell Physiol. 189:266-274.
Langer R, Tirrell DA. 2004. Designing materials for biology and medicine. Nature. 428:487-492.
Lee V, Cao L, Zhang Y, Kiani C, Adams ME, Yang BB. 2000. The roles of matrix molecules in mediating chondrocyte aggregation, attachment, and spreading. J Cell Biochem. 79:322-333.
Li WJ, Danielson KG, Alexander PG, Tuan RS. 2003. Biological response of chondrocytes cultured in three-dimensional nanofibrous poly(epsilon-caprolactone) scaffolds. J Biomed Mater Res A. 67:1105-1114.
Lutolf MP, Hubbell JA. 2005. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol. 23:47-55.
Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, Freed LE, Vunjak-Novakovic G. 2000. Modulation of the mechanical properties of tissue engineered cartilage. Biorheology. 37:141-147.
Martin JA, Buckwalter JA. 2000. The role of chondrocyte-matrix interactions in maintaining and repairing articular cartilage. Biorheology. 37:129-140.
McCarthy JB, Skubitz AP, Qi Z, Yi XY, Mickelson DJ, Klein DJ, Furcht LT. 1990. RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities. J Cell Biol. 110:777-787.
Middleton JC, Tipton AJ. 2000. Synthetic biodegradable polymers as orthopaedic devices. Biomaterials. 21:2335-2346.
Mosher DF, Fogerty FJ, Chernousov MA, Barry EL. 1991. Assembly of fibronectin into extracellular matrix. Ann N Y Acad Sci. 614:167-180.
Mostafavi-Pour Z, Ascari JA, Parkinson SJ, Parker PJ, Ng TT, Humpreies MJ. 2003. Integrin-specific signaling pathways controlling focal adhesion formation and cell migration. J Cell Biol. 161:155-167.
Orend G, Chiquet-Ehrismann R. 2000. Adhesion modulation by antiadhesive molecules of the extracellular matrix. Exp Cell Res. 261:104-110.
Overstreet M, Sohrabi A, Polotsky A, Hungerford DS, Frondoza CG. 2003. Collagen microcarrier spinner culture promotes osteoblast proliferation and synthesis of matrix proteins. In Vitro Cell Dev Biol Anim. 39:228-234.
Pankov R, Yamada KM. 2002. Fibronectin at a glance. J Cell Sci. 115:3861-3863.
Pfaff M. 1997. Recognition sites of RGD-dependent integrins. In: Eble JA, editor. Integrin-Ligand Interaction. Heidelberg: Springer-Verlag. p. 101-121.
Pierschbacher MD, Ruoslahti E. 1984. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 309:30-33.
Rosso F, Giordano A, Barbarisi M, Barbarisi A. 2004. From cell-ECM interactions to tissue engineering. J Cell Physiol. 199:174-180.
Ruoslahti E. 1989. Proteoglycans in cell regulation. J Biol. Chem. 264:13369-13372.
Sakiyama SE, Schense JC, Hubbell JA. 1999. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. FASEB J. 13:2214-2224.
Salgado AJ, Coutinho OP, Reis RL. 2004. Bone tissue engineering: state of the art and future trends. Macromol Biosci. 4:743-765.
Schaffner P, Dard MM. 2003. Structure and function of RGD peptides involved in bone biology. Cell Mol Life Sci. 60:119-132.
Seeger JM, Klingman N. 1985. Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts. J Surg Res. 38:641-647.
Shikani AH, Fink DJ, Sohrabi A, Phan P, Polotsky A, Hungerford DS, Frondoza CG. 2004. Propagation of human nasal chondrocytes in microcarrier spinner culture. Am J Rhinol. 18:105-112.
Shin H, Jo S, Mikos AG. 2003. Biomimetic materials for tissue engineering. Biomaterials. 24:4353-4364.
Slivka MA, Leatherbury NC, Kieswetter K, Niederauer GG. 2000. In vitro compression testing of fiberreinforced, bioabsorbable, porous implants. In: Agrawal CM, Parr JE, Lin ST, eds. Synthetic Bioabsorbable Polymers for Implants, ASTM STP 1396. West Conshohocken, PA: American Society for Testing and Materials. p. 124–135.
Storey RF, Taylor AE. 1998. Effect of stannous octoate on the composition, molecular weight, and molecular weight distribution of ethyleneglycol-initiated poly(ecaprolactone). J Macromol Sci- Pure Appl Chem. A35:723-750.
Stupack DG, Puente XS, Boutsaboualoy S, Storgard CM, Cheresh DA. 2001. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J Cell Biol. 155:459-470.
Tang JS, Chao CF, Au MK. 1994. Growth and metabolism of cultured bone cells using microcarrier and monolayer techniques. Clin Orthop Relat Res. 300:254-258.
Thomson RC, Wake MC, Yaszemski, Mikos AG. 1995. Biodegradable polymer scaffolds to regenerate organs. Adv Polymer Sci. 122:245-274.
Tsai WB, Wang MC. 2005. Effects of an avidin-biotin binding system on chondrocyte adhesion and growth on biodegradable polymers. Macromol Biosci. 5:214-221.
van Hest JC, Tirrell DA. 2001. Protein-based materials, toward a new level of structural control.
Chem Commun. 19:1897-1904.
van Wezel AL. 1967. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature. 216:64-65.
Vunjak-Novakovic G, Martin I, Obradovic B, Treppo S, Grodzinsky AJ, Langer R, Freed LE. 1998. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue engineered cartilage. J Orthop Res. 17:130–138.
Wiesmann HP, Joos U, Meyer U. 2004. Biological and biophysical principles in extracorporal bone tissue engineering. Part II. Int J Oral Maxillofac Surg. 33:523-530.
Williams JM, Adewunmi A, Schek RM, Flanagan CL, Krebsbach PH, Feinberg SE, Hollister SJ, Das S. 2005. Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering. Biomaterials. 26:4817-4827.
Wu SC, Chiang JR, Lin CW. 2004. Novel cell adhesive glycosaminoglycan- binding proteins of Japanese encephalitis virus. Biomacromolecules. 5:2160-2164.
Yokosaki Y, Matsuura N, Higashiyama S, Murakami I, Obara M, Yamakido M, Shigeto N, Chen J, Sheppard D. 1998. Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C. J Biol Chem. 273:11423-11428.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)
全文公開日期本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)

簡易檢索 / 詳目顯示

相關論文