細胞治療在組織工程與再生醫學領域上，為一相當具有前瞻性的治療方法。曾有研究群利用注射幹細胞的方式進行組織再生治療，但效果有限。其原因為單顆懸浮式的細胞在注射過程中，會有大量細胞流失的現象，且植入的細胞會分散於組織各處，使其治療效果受到限制。在實驗室過去的研究裡，已開發出細胞球體培養系統，並利用此系統製備出大小均一之間葉幹細胞(mesnechymal stem cell, MSC)球體。實驗結果顯示，該細胞球體具有完整的細胞外間質及黏附性蛋白，並在注射後可嵌入肌肉間隙中，黏附於組織內部，故可有效提高移植細胞留存於患部的比例。在本論文中，我們使用上述之細胞球體培養系統，結合人類臍帶靜脈內皮細胞(human umbilical vein endothelial cell, HUVEC)與人類臍帶血間葉幹細胞(cord-blood MSC, cbMSC)，製備兩者均勻混和之細胞球體，並將其應用於促進缺血組織血管新生之研究。另一方面，以體外培養方式建立細胞球體時，其內部的細胞可能會因為缺氧而活化低氧誘導因子(hypoxia-inducible factor)及其他與血管新生有關的生長因子。因此我們推測，製備特定大小與細胞密度之球體，使其內部細胞有適當程度的缺氧，能夠讓該球體在植入體內後具有更高的血管新生誘導能力。在體外實驗中，我們製備出均勻混合之HUVEC/cbMSC細胞球體，針對球體大小、細胞密度與缺氧情形等條件進行最佳化，並分析血管結構基因的表現量變化。實驗結果顯示，以10000 cells/well in 85 rpm之條件製備出的HUVEC/cbMSC細胞球體具有最佳的誘導血管新生潛力，因此我們以此條件進行後續的實驗。在體內實驗部分，我們將小鼠股動脈結紮，建立下肢缺血的動物模式後，再將內部處於缺氧狀態之HUVEC/cbMSC細胞球體注射至缺血組織周圍，藉由單光子放射電腦斷層掃描評估治療前後下肢血液灌流的變化情形，並在細胞移植後第十四天進行取樣，實施組織病理切片與免疫染色，評估細胞球體改善組織缺血的能力。動物實驗結果顯示，注射內部細胞有適當缺氧的cbMSC/HUVEC細胞球體可以有效誘導缺血組織的血管新生，改善患部的血液灌流情況，減緩小鼠下肢的萎縮現象。

致謝................................................................................................................I
摘要.............................................................................................................. II
目錄............................................................................................................. III
圖索引........................................................................................................ III
第一章 緒論..................................................................................................1
1.1肢體缺血...........................................................................................1
1.2 細胞移植治療..................................................................................1
1.3 細胞移植治療面臨的難題.............................................................2
1.4 甲基纖維素......................................................................................3
1.5甲基纖維素成膠機制................................................................3
1.6 細胞球體..........................................................................................4
1.7研究動機與實驗目的.......................................................................5
第二章 體外實驗 .......................................................................................10
2.1 研究目的........................................................................................10
2.2 材料與方法....................................................................................10
2.2.1 細胞培養..............................................................................10
2.2.2 甲基纖維素水膠製備.......................................................... 11
2.2.3 細胞球體生產系統建立...................................................... 11
2.2.4含有cbMSC與HUVEC之細胞球體製備方法................12
2.2.5細胞存活率分析...................................................................12
2.2.6 管狀形成實驗(Tube Formation Assay) ..............................13
2.2.7 免疫螢光染色......................................................................13
2.2.8細胞取樣...............................................................................14
2.2.9即時定量連鎖聚合酶反應(Real-Time Quantitative Polymerase Chain Reaction, qPCR) ..............................................14
2.3 實驗結果與討論............................................................................16
2.3.1 細胞球體型態及內部細胞缺氧情形分析.........................16
2.3.2 細胞球體製備條件最佳化..................................................17
2.3.3 細胞球體特性鑑定..............................................................21
2.3.4 細胞存活率分析..................................................................22
2.3.5 生長因子定量分析..............................................................23
2.3.6 類血管網路形成實驗(Tube Formation Assay) ..................24
2.3.7 細胞球體網狀結構特性鑑定..............................................26
2.3.8 細胞球體網狀結構qPCR定量分析................................27
2.4 結論..............................................................................................29
第三章 體內實驗 .......................................................................................30
3.1 研究目的........................................................................................30
3.2 材料與方法....................................................................................30
3.2.1 小鼠下肢缺血的建立..........................................................30
3.2.2單光子放射電腦斷層掃描(single-photon emission computed tomography, SPECT) ..................................................31
3.2.3 病理組織病理分析與免疫染色.........................................32
3.2.4微血管數量測量...................................................................33
3.3實驗結果與討論.............................................................................34
3.3.1 小鼠下肢缺血情形觀察......................................................34
3.3.2微血管數測量.......................................................................36
3.3.3組織切片免疫螢光染色.......................................................37
3.4 結論................................................................................................38
參考文獻......................................................................................................39

1. Rakesh K Jain. Molecular regulation of vessel maturation. Nat Med. 2003;9:685-93.
2. Jain RK, Au P, Tam J, Duda DG, Fukumura D. Engineering vascularized tissue. Nat Biotechol. 2005;23:821-3
3. Segers VF, Revin V, Wu W, Qiu H, Yan Z, Lee RT, et al. Protease-resistant stromal cell-derived factor-1 for the treatment of experimental peripheral artery disease. Circulation. 2011;123:1306-15.
4. Rey S, Lee K, Wang CJ, Gupta K, Chen S, McMillan A, et al. Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated bone marrow-derived angiognetic cells in a mouse model of limb ischemia. Proc Natl Acad Sci USA. 2009; 106:20399-404.
5. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7.
6. Lees AJ, Hardy J, Revesz T. Parkinson's disease. Lancet, 2009;373:2055–2066.
7. Horie M, Sekiya I, Muneta T, et al. Intra-articular Injected synovial stem cells differentiate into meniscal cells directly and promote meniscal regeneration without mobilization to distant organs in rat massive meniscal defect. Stem Cells. 2009;27:878–87.
8. Bhang SH, Cho SW, La WG, et al. Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells. Biomaterials. 2011;32:2734-2747
9. Amann B, Lüdemann C, Rückert R, et al. Design and rationale of a randomized, double-blind, placebo-controlled phase III study for autologous bone marrow cell transplantation in critical limb ischemia: the BONe Marrow Outcomes Trial in Critical Limb Ischemia (BONMOT-CLI) Vasa. 2008;37:319-25.
10. Amann B, Luedemann C, Ratei R, Schmidt-Lucke JA. Autologous bone marrow cell transplantation increases leg perfusion and reduces amputations in patients with advanced critical limb ischemia due to peripheral artery disease. Cell Transplant. 2009;18:371-80.
11. Weck M, Slesaczeck T, Rietzsch H, et al. Noninvasive management of the diabetic foot with critical limb ischemia: current options and future perspectives. Ther Adv Endocrinol Metab. 2011;2:247-55
12. Rosenstrauch D, Poglajen G, Zidar N, Gregoric ID. Stem celltherapy for ischemic heart failure. Tex Heart Inst J. 2005;32:339–47.
13. Fukuda K. Progress in myocardial regeneration and cell transplantation. Circ J. 2005;69:1431-46.
14. Teng CJ, Luo J, Chiu RC, Shum-Tim D. Massive mechanical loss of microspheres with direct intramyocardial injection in the beating heart: implications for cellular cardiomyoplasty. J Thorac Cardiovasc Surg. 2006;132:628-32.
15. Hsieh PC, Davis ME, Gannon J, MacGillivray C, Lee RT. Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest. 2006;116:237-48.
16. Grafflin MW, Derivatives of Cellulose, Cellulose and Cellulose Derivatives, 1963 930-937.
17. Ron E, Bromberg L. Temperature-responsive gels and thermogelling polymer matrices for protein and peptide delivery. Adv Drug Deliv Rev. 1998;31:197–221.
18. Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews. 2001;53:321-39.
19. Haque A, Richardson RK, Morris ER, et al. Thermogelation of methylcellulose: 2 effect of hydroxypropyl substituents. Carbohydrate Polymers 1993;22:175-86
20. Jeong B, Kim SW, Bae YH. Thermosensitive sol-gel reversible hydrogels. Advanced Drug Delivery Reviews. 2002;54:37-51.
21. Chen CH, Tsai CC, Chen W, et al. Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels. Biomacromolecules. 2006;7:736–43.
22. Yang MJ, Chen CH, Lin PJ, Huang CH, Chen W, Sung HW. Novel method of forming human embryoid bodies in a polystyrene dish surface-coated with a temperature-responsive methylcellulose hydrogel. Biomacromolecules. 2007;8:2746-52.
23. Wang CC, Chen CH, Hwang SM, et al. Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty. Stem Cells. 2009;27:724–32.
24. Lee WY, Chang YH, Yeh YC, et al. The use of injectable spherically symmetric cell aggregates self-assembled in a thermo-responsive hydrogel for enhanced cell transplantation. Biomaterials. 2009;30:5505–13.
25. Lee WY, Wei HJ, Lin WW, et al. Enhancement of cell retention and functional benefits in myocardial infarction using human amniotic-fluid stem-cell bodies enriched with endogenous ECM. Biomaterials. 2011;32:5558–5567.
26. Staton CA, Stribbling SM, Tazzyman S, Hughes R, Brown NJ, Lewis CE. Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Patho. 2004;85:233-48.
27. Kubota Y, Kleinman HK, Martin GR, Lawley TJ, Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol. 1998;107:1589-98.
28. Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK. Tissue engineering: creation of long-lasting blood vessels. Nature. 2004;428:138-9.
29. Nör JE, Peters MC, Christensen JB, et al. Engineering and characterization of functional human microvessels in immunodeficient mice. Lab Invest. 2001;81:453-63
30. Traktuev DO, Prater DN, Merfeld-Clauss S, et al. Robust functional vascular network formation in vivo by cooperation of adipose progenitor and endothelial cells. Circ Res. 2009;104:1410–20.
31. Au P, Tam J, Fukumura D, Jain RK. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood. 2008;111:4551-58.
32. Jain RK. Molecular regulation of vessel maturation Nat Med. 2008;9:685-93.
33. Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol. 1998;141:805-14.
34. Melero-Martin JM, De Obaldia ME, Kang SY et al. Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells. Circ Res. 2008;103:194-202.
35. Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev. 2011;63:300–11.
36. Rouwkema J, Rivron NC, van Blitterswijk CA. Vascularization in tissue engineering. Trends Biotechnol. 2008;26:434–41.
37. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003;9:677–84.
38. Rey S, Lee K, Wang CJ, et al. Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia. Proc Natl Acad Sci USA. 2009;106:20399-404.
39. Liao YH, Xia N, et al. Interleukin-17A Contributes to Myocardial Ischemia/Reperfusion Injury by Regulating Cardiomyocyte Apoptosis and Neutrophil Infiltration. J Am Coll Cardiol. 2012;59:420–29.
40. Cui LY, Yang S, Zhang J. Protective Effects of Neutrophil Gelatinase–Associated Lipocalin on Hypoxia/Reoxygenation Injury of HK-2 Cells. Transplant Proc. 2011;43:3622-7.
41. Hwang NS, Varghese S, Elisseeff J. Controlled differentiation of stem cells. Adv Drug Deliv Rev. 2008;60:199–214.
42. Chen S, Fitzgerald W, Zimmerberg J, et al. Cell-cell and cell-extracellular matrix interactions regulate embryonic stem cell differentiation. Stem Cells. 2007;25:553–61.
43. Neff JA, Tresco PA, Caldwell KD. Surface modification for controlled studies of cell-ligand interactions. Biomaterials, 1999;20:2377–93.
44. Delafontaine P, Song YH, Li Y. Expression, regulation, and function of IGF-1, IGF-1R, and IGF-1 binding proteins in blood vessels. Arterioscler Thromb Vasc Biol. 2004;24:435-44.
45. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008;103:1204-19.
46. Jeon O, Kang SW, Lim HW, et al. Synergistic effect of sustained delivery of basic fibroblast growth factor and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs. Biomaterials. 2006;27:1617-25.