結合抗氧化劑與可注射式幹細胞球體之複合式療法於缺血性疾病治療之應用

A Combination Strategy Using An Antioxidant and Injectable Stem Cell Aggregates for Treating Ischemic Diseases

黃玠誠(Chieh-Cheng Huang)

指導教授 ： 宋信文

國立清華大學/工學院/化學工程學系/博士(2015年)

摘要

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。其原因在於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，且植入的細胞過於分散在組織各處，使其治療效果受到限制。此外，缺血組織內部會因發炎反應而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。本實驗室過去的研究中，已開發出細胞球體培養系統，並利用此系統培養出大小均一之間葉幹細胞(mesenchymal stem cell, MSC)球體，且其體積足以鑲嵌在肌肉間隙中，避免細胞流失。實驗結果顯示，該細胞球體具有較大的體積與完整的細胞外間質結構，因此移植後留存於注射部位的細胞數量遠多於單顆懸浮式的細胞。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三維細胞球體，並將其應用於促進缺血組織血管新生之研究。另一方面，細胞球體內部的細胞可能會因缺氧而活化低氧誘導因子及其他生長因子，使該細胞球體具有更高的血管新生誘導能力。在本論文的第一部分裡，我們發現HUVEC/cbMSC球體在Matrigel表面形成管狀結構時，會表現大量的血管新生標記αVβ3 integrin，並分泌多種生長因子，證明其具有促進血管新生之潛能。在動物實驗中，我們將HUVEC/cbMSC細胞球體注射至大鼠心肌梗塞病灶處，再分別以正子放射造影、單光子放射電腦斷層掃描與心臟超音波觀察梗塞心肌的血管新生、血液灌流變化與心室功能的恢復情況。實驗結果顯示，移植HUVEC/cbMSC細胞球體可以有效促進梗塞部位的血管新生，使得缺血組織的血液灌流恢復，進而促進心室功能的改善。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，在本論文的第二部分中，我們使用抗氧化劑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。體外實驗結果顯示，NAC可以有效降低過氧化氫的氧化力，使細胞球體於氧化壓力下的貼附、生長與血管新生能力得以維持。在動物實驗部分，我們將HUVEC/cbMSC細胞球體懸浮於含有NAC的食鹽水中，並注射至以手術方式建立的小鼠左下肢缺血部位。實驗結果顯示，NAC可有效降低缺血組織內的氧化壓力，提升植入細胞的留存率。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。由以上實驗結果可知，內部缺氧之HUVEC/cbMSC細胞球體可以有效地促進缺血組織的血管新生；而結合抗氧化劑NAC與細胞球體之複合式療法則能更進一步提升細胞移植後的留存率與治療效果，未來或有應用於缺血性疾病治療的潛能。

關鍵字

細胞療法 ； 缺血性疾病 ； 血管新生 ； 抗氧化劑 ； 活性氧化物質

並列摘要

A recurring obstacle for cell-base strategies in treating ischemic diseases continues to be the significant cell loss during the process of transplantation. Additionally, the recipient microenvironment in the ischemic tissues may confer an elevated state of oxidative stress to the administered cells, thus hindering their adhesion and retention to the therapeutic target, ultimately limiting the scope of therapeutic benefit. In our previous studies, a thermo-responsive methylcellulose (MC) hydrogel system was employed to grow three-dimensional cell aggregates for the treatment of ischemic diseases. By using human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs), the fabricated cell aggregates have great potential in inducing therapeutic angiogenesis. Using a rat model of myocardial infarction (MI), the cell aggregates that were transplanted intramuscularly via local injection were demonstrated to be entrapped effectively in the interstices of muscular tissues. The engrafted cells subsequently promoted considerable angiogenesis, improving the post-infarcted heart function. Although the therapeutic efficacy of HUVEC/cbMSC aggregates appears to be favorable, the mechanism of their angiogenesis in repairing ischemic tissues remains elusive. In Study I, the process of cell-mediated angiogenesis and its therapeutic effects that are induced by exogenously engrafted HUVEC/cbMSC aggregates in rats with MI are investigated. By maximizing cell‒cell and cell‒ECM communications and establishing a hypoxic microenvironment in their inner cores, these cell aggregates are capable of forming widespread tubular networks together with the angiogenic marker αvβ3 integrin when grown on Matrigel. The aggregates of HUVECs/cbMSCs are exogenously engrafted into the peri-infarct zones of rats with MI via direct local injection. Multimodality noninvasive imaging techniques, including positron emission tomography, single photon emission computed tomography, and echocardiography, are employed to monitor serially the beneficial effects of cell therapy on angiogenesis, blood perfusion, and global/regional ventricular function, respectively. The myocardial perfusion is correlated with ventricular contractility, demonstrating that the recovery of blood perfusion helps to restore regional cardiac function, leading to the improvement in global ventricular performance. These experimental data reveal the efficacy of the exogenous transplantation of 3D cell aggregates after MI and elucidate the mechanism of cell-mediated therapeutic angiogenesis for cardiac repair. In Study II, we hypothesize that by concurrent delivery of an antioxidant N-acetylcysteine (NAC), the cell retention following transplantation of HUVEC/cbMSC aggregates in a mouse model with hindlimb ischemia may be significantly augmented. Our in vitro results demonstrate that the antioxidant NAC can successfully restore the reactive oxygen species (ROS)-impaired cell adhesion and recover the reduced angiogenic potential of HUVEC/cbMSC aggregates. In the animal study, we found that by scavenging the ROS generated in ischemic tissues, NAC has great potential to establish a receptive cell environment at the early stage of cell transplantation, thereby promoting the cell adhesion, retention, and survival of engrafted cell aggregates, which subsequently enhances therapeutic angiogenesis and ultimately results in blood flow recovery and limb salvage. The combinatory strategy using an antioxidant and cell aggregates may offer a new opportunity to boost the therapeutic efficacy for the treatment of ischemic diseases.

並列關鍵字

cell therapy ； ischemic diseases ； angiogenesis ； antioxidant ； reactive oxygen species

參考文獻

[1] Laflamme MA, Murry CE. Heart regeneration. Nature 2011;473:326–35.

[2] Dolgin E. Taking tissue engineering to heart. Nat Med 2011;17:1032–5.

[3] Widmer LK, Greensher A, Kannel WB. Occlusion of Peripheral Arteries: A Study of 6,400 Working Subjects. Circulation 1964;30:836–52.

[4] Segers VFM, Revin V, Wu W, Qiu H, Yan Z, Lee RT, Sandrasagra A. Protease-resistant stromal cell-derived factor-1 for the treatment of experimental peripheral artery disease. Circulation 2011;123:1306–15.

[5] Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005;23:845–56.