以大鼠心肌梗塞模型評估內部處於缺氧狀態之 三維細胞球體血管新生及恢復心肌功能的可行性

Hypoxia-induced Therapeutic Angiogenesis in a Rat Model of Myocardial Infarction Using Injectable 3D Cell Aggregates

王靖雯(Wang, Ching Wen)

指導教授 ： 宋信文

國立清華大學/工學院/生物醫學工程研究所/碩士(2015年)

摘要

成人的心肌組織缺乏再生能力，梗塞部位會因為缺血壞死而逐漸纖維化，進而影響心室功能。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。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，該細胞球體具有完整的細胞外間質結構及黏附性蛋白，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，故對梗塞後的心臟功能改善有明顯的作用。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，並將其應用於促進梗塞組織血管新生與心臟功能修復之研究。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(hypoxia-inducible factor, HIF)及其他與血管新生相關的生長因子；因此藉由製備特定大小與細胞密度之細胞球體，使其內部呈現部分缺氧狀態，能夠讓該細胞球體具有更高的血管新生誘導能力。在體外實驗中，我們製備出內部缺氧之HUVEC/cbMSC細胞球體，並以管狀形成實驗評估其血管新生潛能。實驗結果顯示，HUVEC/cbMSC球體在形成管狀結構時，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。在動物實驗部分，我們以外科手術建立大鼠心肌梗塞模式，將內部缺氧之HUVEC/cbMSC細胞球體注射至梗塞組織周圍後，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。我們發現移植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。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，未來或許可應用於心肌梗塞或其他缺血性疾病的細胞治療。

關鍵字

細胞療法缺氧 ； 血管新生 ； 缺氧 ； 缺血性疾病 ； 心肌梗塞

並列摘要

Cell transplantation via direct intramyocardial injection is a promising therapy for patients with myocardial infarction (MI). Following intramuscular injection, however, retention of these dissociated cells at the cell graft site remains problematic; this poor retention adversely affects the efficacy of cell-transplantation therapy. In our previous study, a method for constructing spherically symmetric three-dimensional (3D) cell aggregates was developed by using a thermos-responsive methylcellulose (MC) hydrogel system. The grown cell aggregates can be harvested without using proteolytic enzymes; consequently, their inherent extracellular matrices (ECMs) and integrative adhesive agents remain well preserved. With an adequate physical size and adhesive ECMs, the 3D cell aggregates can entrap and retain in the muscular interstices after transplantation. Therefore, the remaining amount of transplanted cells is more than their dissociated counterparts. On the other hand, cell aggregates can develop a hypoxic microenvironment in their inner cores at distances that exceed the diffusion capacity of oxygen. By switching on a series of signal transduction mechanisms, hypoxia-inducible factors (HIFs) can cause the transcriptional activation of several pro-angiogenic genes. As a result, we hypothesize that transplantation of internally-hypoxia cell aggregates can trigger robust angiogenesis by HIF-1α-dependent angiogenic mechanisms prior to cell engraftment, thereby enhancing regional blood perfusion. In this work, 3D aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) are constructed using the methylcellulose hydrogel system. These cell aggregates are capable of forming widespread tubular networks together with the angiogenic marker αvβ3 integrin; they secret multiple pro-angiogenic, pro-survival, and mobilizing factors 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 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.

並列關鍵字

cell-based therapy ； angiogenesis ； hypoxia ； ischemic diseases ； myocardial infarction

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