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針對缺血性疾病進行治療，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；此外，缺血組織內部發炎反應會產生大量的活性氧化物質(reactive oxygen species, ROS)，傷害細胞中的核酸、蛋白質與脂質，使植入體內的細胞不易貼附生長而發揮治療效果。在實驗室過去的研究裡，已成功開發出細胞球體培養系統，並利用此系統製備出含有人類臍帶靜脈內皮細胞(human umbilical vein endothelial cell, HUVEC)與臍帶血間葉幹細胞(cord-blood mesnechymal stem cell, cbMSC)之三維球體。實驗結果顯示，該細胞球體可以有效提升細胞移植後的留存率，並幫助缺血組織產生血管新生。為了進一步提升移植細胞的貼附能力及其後續的治療效果，本論文使用抗氧化劑N-乙醯半胱氨酸(N-acetylcysteine, NAC)與細胞球體一起進行注射，期望藉由NAC的抗氧化功能降低缺血組織內的氧化壓力。在體外實驗中，我們發現NAC可以有效降低過氧化氫的氧化力，並證明NAC可以使細胞球體於氧化壓力下的貼附能力顯著增加，使細胞能夠正常生長，並分泌血管新生相關的生長因子。在體內實驗部分，我們以手術方式將小鼠左腿股動脈結紮，建立下肢缺血的模式後，再將HUVEC/cbMSC細胞球體懸浮於含有NAC的食鹽水中，並注射至缺血組織周圍；實驗中，以僅注射生理食鹽水、僅注射NAC與僅注射HUVEC/cbMSC球體等三個組別做為控制組。動物實驗結果顯示，NAC可有效降低缺血組織內的氧化壓力，提升細胞留存率。而從單光子放射電腦斷層掃描與組織免疫染色的結果中，我們發現同時注射NAC與細胞球體可以有效誘導組織血管新生，改善患部血液灌流情況，減緩小鼠下肢萎縮。此外，我們亦使用即時定量聚合酶連鎖反應針對人類特有的基因序列進行分析，發現NAC確實可以增加留存於組織中的人類細胞。以上實驗結果證實抗氧化劑NAC能夠減少缺血組織內的氧化壓力，進而提升細胞移植後的存留率與其治療效果。

Cell transplantation for therapeutic neovascularization holds great promise for treating ischemic diseases. However, following injections, retention of transplanted cells in engrafted areas remains problematic, and can be deleterious to cell-transplantation therapy. Additionally, reactive oxygen species (ROSs) are generated during tissue ischemia, which may disrupt cellular function of the engrafted cells via oxidation of major cellular macromolecules, such as lipid, DNA and protein. To enhance the cell retention and the subsequent therapeutic benefits, three-dimensional aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) that had been reported in our previous studies were concurrently delivered with an antioxidant N-acetylcysteine (NAC) for the treatment of ischemic disease. The results of in vitro studies demonstrated that NAC can effectively reduce the environmental oxidative stress and improve the cell adhesion ability. Transplantation of HUVEC/cbMSC aggregates with NAC into a mouse model of an ischemic limb significantly reduced regional oxidative stress, promoted formation of functional vessels, improved limb blood perfusion, and attenuated muscle atrophy and bone losses, thereby rescuing tissue degeneration. Notably, the retention of engrafted cells and their therapeutic efficacy were significantly improved by the concurrent delivery of NAC. These analytical results demonstrate that by co-delivery with antioxidant NAC, the potential of HUVEC/cbMSC aggregates for therapeutic neovascularization can be markedly enhanced.

摘要 I
圖錄 V
第一章 緒論 1
1.1下肢缺血 1
1.2缺血組織內的活性氧化物質(reactive oxygen species, ROS) 1
1.3 細胞移植治療 3
1.4 細胞移植治療面臨的難題 4
1.4.1 移植後的細胞留存率不足 4
1.4.2缺血組織內的高氧化壓力 4
1.5 抗氧化劑N-乙醯半胱胺酸(N-acetylcysteine, NAC) 5
1.6 研究動機與實驗目的 5
第二章 材料與方法 7
2.1細胞培養 7
2.2 甲基纖維素水膠製備 7
2.3 細胞球體生產系統建立 7
2.4含有cbMSC與HUVEC之細胞球體製備方法 8
2.5細胞毒性分析 9
2.6細胞貼附實驗 9
2.7 Live/Dead染色 9
2.8管狀形成實驗(Tube Formation Assay) 9
2.9生長因子定量分析 10
2.10免疫螢光染色 10
2.11手術程序 10
2.12單光子放射電腦斷層掃描 (single-photon emission computed tomography, SPECT) 11
2.13 脂質過氧化分析 11
2.14組織DNA萃取與即時定量連鎖聚合酶反應(real-time quantitative polymerase chain reaction, qPCR) 11
2.15病理組織病理分析與免疫染色 12
第三章 實驗結果 13
3.1 體外實驗 13
3.1.1 NAC對於cbMSC和HUVEC之細胞毒性分析 13
3.1.2 NAC之抗氧化能力 13
3.1.3 NAC增加細胞在氧化壓力下的存活、貼附與生長能力 14
3.1.4血管新生潛能評估 17
3.2動物實驗 19
3.2.1 短期內組織氧化壓力傷害 19
3.2.2小鼠缺血下肢血液灌流恢復情形觀察 21
3.2.3 血管數觀察 23
3.2.4細胞球移植後於組織內的存活率 24
第四章 結論 26
參考資料 276

1. Jain, R.K., Molecular regulation of vessel maturation. Nat Med, 2003. 9(6): p. 685-93.
2. Jain, R.K., et al., Engineering vascularized tissue. Nat Biotechnol, 2005. 23(7): p. 821-3.
3. Segers, V.F., et al., Protease-resistant stromal cell-derived factor-1 for the treatment of experimental peripheral artery disease. Circulation, 2011. 123(12): p. 1306-15.
4. Rey, S., 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 U S A, 2009. 106(48): p. 20399-404.
5. Droge, W., Free radicals in the physiological control of cell function. Physiol Rev, 2002. 82(1): p. 47-95.
6. Toledano, B.D.A.a.M.B., ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Natural Review Molecular Cell Biology.
7. Circu, M.L. and T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med, 2010. 48(6): p. 749-62.
8. Thannickal, V.J. and B.L. Fanburg, Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol, 2000. 279(6): p. L1005-28.
9. Lambeth, J.D., NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol, 2004. 4(3): p. 181-9.
10. Mittal, M., et al., Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal, 2014. 20(7): p. 1126-67.
11. Frangogiannis, N.G., C.W. Smith, and M.L. Entman, The inflammatory response in myocardial infarction. Cardiovasc Res, 2002. 53(1): p. 31-47.
12. Vergely, C., et al., Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy. Arch Biochem Biophys, 2003. 420(2): p. 209-16.
13. Nahrendorf, M., M.J. Pittet, and F.K. Swirski, Monocytes: protagonists of infarct inflammation and repair after myocardial infarction. Circulation, 2010. 121(22): p. 2437-45.
14. Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem cells. Science, 1999. 284(5411): p. 143-7.
15. Horie, M., 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(4): p. 878-87.
16. Bhang, S.H., et al., Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells. Biomaterials, 2011. 32(11): p. 2734-47.
17. Amann, B., 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(4): p. 319-25.
18. Amann, B., et al., 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(3): p. 371-80.
19. Drowley, L., et al., Cellular antioxidant levels influence muscle stem cell therapy. Mol Ther, 2010. 18(10): p. 1865-73.
20. Rahman, I., Pharmacological antioxidant strategies as therapeutic interventions for COPD. Biochim Biophys Acta, 2012. 1822(5): p. 714-28.
21. Chen, W., et al., A redox-based mechanism for cardioprotection induced by ischemic preconditioning in perfused rat heart. Circ Res, 1995. 77(2): p. 424-9.
22. Ferrari, R., et al., Oxygen free radicals and myocardial damage: protective role of thiol-containing agents. Am J Med, 1991. 91(3C): p. 95S-105S.
23. Lee, W.Y., 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(24): p. 5558-67.
24. Huang, C.C., et al., Hypoxia-induced therapeutic neovascularization in a mouse model of an ischemic limb using cell aggregates composed of HUVECs and cbMSCs. Biomaterials, 2013. 34(37): p. 9441-50.
25. Laflamme, M.A., et al., Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol, 2007. 25(9): p. 1015-24.