臺灣博碩士論文加值系統

隨著組織工程研究的蓬勃發展，對於幫助組織或器官之修復被視為重要課題之一，且新的組織生長與發育需要適當的血管用以傳送養分以及移除廢物，而目前許多研究發現許多血管新生生長因子具有幫助血管新生、組織修復的功能，因此，尋找適當的血管新生生長因子，以體外培養方式，來進行血管新生之實驗。本論文希望瞭解4種血管新生生長因子及其混合液的功能，進一步結合實驗室之前所研發之多孔狀膠原蛋白基質，進行血管新生之試驗。於實驗發現添加生長因子於二維培養下，對於細胞活性有顯著之影響，而於細胞爬行實驗上，會隨著不同生長因子、濃度及細胞而有所差異。因此進一步以三維立體培養方式進行共同培養內皮細胞及纖維母細胞於多孔狀膠原蛋白基質中，來評估並比較生長因子的添加對於細胞增生及血管新生之影響，於實驗發現添加生長因子於多孔狀膠原蛋白基質中的確可促進血管新生，且進一步利用組織免疫染色證實其管狀結構為細胞所構成，且其管狀結構周圍有基底膜生成。

In light of the rapid development of tissue engineering, angiogenesis has become one of the major issues in tissue and organ repair. Since the development of new tissues requires blood vessels to transport nutrient and remove wastes, it was found recently that, some angiogenic factors are capable of facilitating angiogenesis and tissue rehabilitation. Thus, this research aims at the screening of adequate angiogenic factors, and their ability to promote angiogenesis was investigated in an in vitro cell culture system. The functions of 4 various angiogenic factors was also examined. For a three-dimensional culture system, porous collagen matrices previously developed in our laboratory were applied.
It was found in this study that the adding of angiogenic factors under 2-D condition exhibited a significant effect on cell activities. Further, the concentration of angiogenic factors, the cell types, and various combinations of angiogenic factors were shown to affect the behavior of cell migration. Besides, a co-culture system of simultaneously cultivating endothelial cells and fibroblasts in porous collagen matrices were established to evaluate and compare the effect on cell proliferation and angiogenesis. It was found that the existence of angiogenic factors in porous collagen matrices did facilitate angiogenesis. The immunohistochemical staining was further carried out in this study to corroborate the tubular structure which was composed of cells, and basement membrane formation around these tubular structures.

目錄
中文摘要
英文摘要
目錄
表目錄
圖目錄
第一章 緒論…………………………………………………………………1
1.1 膠原蛋白………………………………………………………….……1
1.1.1 膠原蛋白的性質與結構………..…………………………………1
1.1.2 膠原蛋白在醫學上的應用………..………………………………2
1.2 血管新生………………………………………………………….……3
1.2.1 血管新生的過程……………………………………….…….……3
1.2.2 血管新生的影響因素………………………………….…….……5
1.2.3 血管新生的調控機轉………………………………….…….……8
1.2.4 血管新生對於組織工程之重要性…………….……….…….…..10
1.3 血管新生之培養模式……………………………………...…….……11
1.3.1 體外試驗模式…………….…………………………...…….……11
1.3.1.1 二維培養模式………….……………………….…...…….……12
1.3.1.2 三維培養模式………….……………………….…...…….……12
1.3.2 活體實驗模式………….……………………….…...…….…...…13
1.3.2.1 雞胚胎卵黃囊試驗法….…………………….…...……..…...…14
1.3.2.2 眼角膜上血管新生試驗.…………………….…...……..…...…14
1.3.2.3 The Matrigel plug assay.…………………….…...…..…..…...…15
1.3.2.4 Chamber assays.…………………………….…...…..…..…...…15
1.4 研究目的.………………………………………….…...…..…..…...…15
第二章 實驗儀器、藥品、與方法…………………….…...…..…..…...…17
2.1 實驗儀器………………………………….……….…...…..…..…...…17
2.2 實驗藥品………………………………….……….…...…..…..…...…19
2.3 實驗方法………………………………….……….…...…..…..…...…21
2.3.1 添加不同比例濃度之bFGF對於細胞活性之影響.…..…..….…21
2.3.2 添加不同比例濃度之bFGF對於細胞數目之影響.…..…..….…22
2.3.3 添加不同之生長因子對於細胞爬行之影響.……….....…..….…22
2.3.4 PI染色…………….………………….……….…...…..…..…...…23
2.3.5 三維細胞培養…….………………….……….…...…..…..…...…23
2.3.6 基質的包埋與切片.………………….……….…...…..…..…...…23
2.3.6.1 冷凍切片…….………………….……….……....…..…..…...…23
2.3.6.2 塑膠切片…….………………….……….……....…..…..…...…24
2.3.7 基質之組織切片H&E染色……….……….……....…..…..…...24
2.3.7.1 冷凍切片……….……….……………………....…..…..…...…24
2.3.7.2 塑膠切片……….……….……………………....…..…..…...…25
2.3.8 免疫組織化學染色法…….……………………....…..…..…...…25
2.3.9 Masson’s trichrome染色…….……………………....………...…25
第三章 生長因子對於細胞之影響…….……………………....……...…27
3.1 實驗目的…….……………………....……………………………...…27
3.2 實驗材料與方法…………………....……………………………....…27
3.2.1 實驗材料…………………....……..…………………………...…27
3.2.2 添加不同比例濃度之bFGF對於細胞活性之影響…………...…28
3.2.3 添加不同比例濃度之bFGF對於細胞數目之影響…………...…28
3.2.4 添加不同生長因子對於細胞爬行之影響………………..…...…29
3.4 實驗結果…………………....……..………...……………………...…29
3.4.1 添加不同比例濃度之bFGF對於細胞活性之影響…………...…29
3.4.2 添加不同比例濃度之bFGF對於細胞數目之影響…………...…29
3.4.3 添加不同生長因子對於細胞爬行之影響………….……………30
3.5 討論…………………....……..……….……..……………………...…31
第四章 三維立體基質培養.……..……….……..……………………...…32
4.1 實驗目的.……..……………………....……..……………………...…32
4.2 實驗材料與方法……………………....…....……….……………...…32
4.2.1 實驗材料……………………....…....……….……………...…….32
4.2.2 三維細胞培養………………....…....……….……………...…….33
4.2.3 生長因子對於刺激細胞增生之影響..…….………………...…...33
4.2.4 抗原表現情況………………....….....……………………...…….33
4.2.5 血管結構變化………………....….....……………………...…….33
4.2.6 血管管狀型態………………....….....……………………...…….33
4.3 實驗結果…………………………....…....……………………...…….33
4.3.1 生長因子對於刺激細胞增生及管狀生成之影響…………...…..34
4.3.2 生長因子對於抗原表現之情況……………………………...…..34
4.3.3 生長因子對於血管結構之變化……………………………...…..35
4.4 討論………………………………………...……………………...…..35
第五章 實驗結論……….……………………...……………………...…..36
第六章 文獻回顧……….……………………...……………………...…..38

Akhtar, N., E. B. Dickerson, et al. (2002). "The sponge/Matrigel angiogenesis assay." Angiogenesis 5(1-2): 75-80.
Auger, F. A., M. Rouabhia, et al. (1998). "Tissue-engineered human skin substitutes developed from collagen- populated hydrated gels: clinical and fundamental applications." Med Biol Eng Comput 36(6): 801-12.
Augustin, H. G. (1998). "Antiangiogenic tumour therapy: will it work?" Trends Pharmacol Sci 19(6): 216-22.
Ausprunk, D. H. and J. Folkman (1977). "Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis." Microvasc Res 14(1): 53-65.
Bach, T. L., C. Barsigian, et al. (1998). "VE-Cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels." Exp Cell Res 238(2): 324-34.
Banda, M. J., D. R. Knighton, et al. (1982). "Isolation of a nonmitogenic angiogenesis factor from wound fluid." Proc Natl Acad Sci U S A 79(24): 7773-7.
Black, A. F., F. Berthod, et al. (1998). "In vitro reconstruction of a human capillary-like network in a tissue- engineered skin equivalent." Faseb J 12(13): 1331-40.
Brooks, P. C. (1996). "Cell adhesion molecules in angiogenesis." Cancer Metastasis Rev 15(2): 187-94.
Brooks, P. C. (1996). "Role of integrins in angiogenesis." Eur J Cancer 32A(14): 2423-9.
Burgess, B. T., J. L. Myles, et al. (2000). "Quantitative analysis of adhesion-mediated cell migration in three- dimensional gels of RGD-grafted collagen." Ann Biomed Eng 28(1): 110-8.
Butler, D. L. and H. A. Awad (1999). "Perspectives on cell and collagen composites for tendon repair." Clin Orthop(367 Suppl): S324-32.
Caterson, E. J., L. J. Nesti, et al. (2001). "Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/alginate amalgam." J Biomed Mater Res 57(3): 394-403.
Cavender, J. L. and W. J. Murdoch (1988). "Morphological studies of the microcirculatory system of periovulatory ovine follicles." Biol Reprod 39(4): 989-97.
Chang, J. H., E. E. Gabison, et al. (2001). "Corneal neovascularization." Curr Opin Ophthalmol 12(4): 242-9.
Cockerill, G. W., J. R. Gamble, et al. (1995). "Angiogenesis: models and modulators." Int Rev Cytol 159: 113-60.
Conway, E. M., D. Collen, et al. (2001). "Molecular mechanisms of blood vessel growth." Cardiovasc Res 49(3): 507-21.
de Chalain, T., J. H. Phillips, et al. (1999). "Bioengineering of elastic cartilage with aggregated porcine and human auricular chondrocytes and hydrogels containing alginate, collagen, and kappa-elastin." J Biomed Mater Res 44(3): 280-8.
Doillon, C. J., F. H. Silver, et al. (1987). "Fibroblast growth on a porous collagen sponge containing hyaluronic acid and fibronectin." Biomaterials 8(3): 195-200.
Doughty, M. J. (2001). "Changes in hydration, protein and proteoglycan composition of the collagen-keratocyte matrix of the bovine corneal stroma ex vivo in a bicarbonate-mixed salts solution, compared to other solutions." Biochim Biophys Acta 1525(1-2): 97-107.
Dusseau, J. W., P. M. Hutchins, et al. (1986). "Stimulation of angiogenesis by adenosine on the chick chorioallantoic membrane." Circ Res 59(2): 163-70.
Folkman, J. (1985). "Tumor angiogenesis." Adv Cancer Res 43: 175-203.
Folkman, J. (1986). "How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes memorial Award lecture." Cancer Res 46(2): 467-73.
Folkman, J. and Y. Shing (1992). "Angiogenesis." J Biol Chem 267(16): 10931-4.
Freedman, S. B. and J. M. Isner (2002). "Therapeutic angiogenesis for coronary artery disease." Ann Intern Med 136(1): 54-71.
Gaede, S. D., M. M. Sholley, et al. (1985). "Endothelial mitosis during the initial stages of corpus luteum neovascularization in the cycling adult rat." Am J Anat 172(2): 173-80.
Gibran, N. S., F. F. Isik, et al. (1994). "Basic fibroblast growth factor in the early human burn wound." J Surg Res 56(3): 226-34.
Giordano, F. J. (1999). "Angiogenesis: mechanisms, modulation, and targeted imaging." J Nucl Cardiol 6(6): 664-71.
Hanahan, D. and J. Folkman (1996). "Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis." Cell 86(3): 353-64.
Kohn, E. C., R. Alessandro, et al. (1995). "Angiogenesis: role of calcium-mediated signal transduction." Proc Natl Acad Sci U S A 92(5): 1307-11.
Lee, C. H., A. Singla, et al. (2001). "Biomedical applications of collagen." Int J Pharm 221(1-2): 1-22.
Liekens, S., E. De Clercq, et al. (2001). "Angiogenesis: regulators and clinical applications." Biochem Pharmacol 61(3): 253-70.
Ma, J., H. Wang, et al. (2001). "A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts." Biomaterials 22(4): 331-6.
Machens, H. G., A. C. Berger, et al. (2000). "Bioartificial skin." Cells Tissues Organs 167(2-3): 88-94.
Maciag, T. (1984). "Angiogenesis." Prog Hemost Thromb 7: 167-82.
Makris, A., K. J. Ryan, et al. (1984). "The nonluteal porcine ovary as a source of angiogenic activity." Endocrinology 115(5): 1672-7.
Mandriota, S. J., P. A. Menoud, et al. (1996). "Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells." J Biol Chem 271(19): 11500-5.
Matsuda, K., S. Suzuki, et al. (1990). "Influence of glycosaminoglycans on the collagen sponge component of a bilayer artificial skin." Biomaterials 11(5): 351-5.
Morishita, R., S. Nakamura, et al. (1999). "Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy." Hypertension 33(6): 1379-84.
Mosahebi, A., M. Simon, et al. (2001). "A novel use of alginate hydrogel as Schwann cell matrix." Tissue Eng 7(5): 525-34.
Moscatelli, D. A., M. Presta, et al. (1986). "Purification and biological activities of an angiogenesis factor from human placenta." Anticancer Res 6(4): 861-3.
Murray, M. M. and M. Spector (2001). "The migration of cells from the ruptured human anterior cruciate ligament into collagen-glycosaminoglycan regeneration templates in vitro." Biomaterials 22(17): 2393-402.
Norrby, K., A. Jakobsson, et al. (1990). "Increased angiogenesis in diabetes." Experientia 46(8): 856-60.
Norrby, K., A. Jakobsson, et al. (1990). "Quantitative angiogenesis in spreads of intact rat mesenteric windows." Microvasc Res 39(3): 341-8.
Raju, K. S., G. Alessandri, et al. (1984). "Characterization of a chemoattractant for endothelium induced by angiogenesis effectors." Cancer Res 44(4): 1579-84.
Rak, J., J. Filmus, et al. (1996). "Reciprocal paracrine interactions between tumour cells and endothelial cells: the ''angiogenesis progression'' hypothesis." Eur J Cancer 32A(14): 2438-50.
RayChaudhury, A. and P. A. D''Amore (1991). "Endothelial cell regulation by transforming growth factor-beta." J Cell Biochem 47(3): 224-9.
Ribatti, D., A. Gualandris, et al. (1997). "New model for the study of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane: the gelatin sponge/chorioallantoic membrane assay." J Vasc Res 34(6): 455-63.
Ribatti, D., B. Nico, et al. (1997). "Basic fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane: an electron microscopy study." Microvasc Res 53(2): 187-90.
Rifkin, D. B., D. Moscatelli, et al. (1983). "Proteases, angiogenesis, and invasion." Symp Fundam Cancer Res 36: 187-200.
Salcedo, R., K. Wasserman, et al. (1999). "Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: In vivo neovascularization induced by stromal-derived factor-1alpha." Am J Pathol 154(4): 1125-35.
Smith-McCune, K., Y. H. Zhu, et al. (1997). "Cross-species comparison of angiogenesis during the premalignant stages of squamous carcinogenesis in the human cervix and K14-HPV16 transgenic mice." Cancer Res 57(7): 1294-300.
Tonnesen, M. G., X. Feng, et al. (2000). "Angiogenesis in wound healing." J Investig Dermatol Symp Proc 5(1): 40-6.
Velazquez, O. C., R. Snyder, et al. (2002). "Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like 3-dimensional networks." Faseb J 16(10): 1316-8.
West, D. C., I. N. Hampson, et al. (1985). "Angiogenesis induced by degradation products of hyaluronic acid." Science 228(4705): 1324-6.
Xue, L. and H. P. Greisler (2002). "Angiogenic effect of fibroblast growth factor-1 and vascular endothelial growth factor and their synergism in a novel in vitro quantitative fibrin-based 3-dimensional angiogenesis system." Surgery 132(2): 259-67.
Yang, E. K., Y. K. Seo, et al. (2000). "Tissue engineered artificial skin composed of dermis and epidermis." Artif Organs 24(1): 7-17.
Yasuda, Y., Y. Fujita, et al. (2002). "Erythropoietin is involved in growth and angiogenesis in malignant tumours of female reproductive organs." Carcinogenesis 23(11): 1797-805.
Zetter, B. R. (1988). "Angiogenesis. State of the art." Chest 93(3 Suppl): 159S-166S.