細胞治療應用於缺血性心臟疾病之效果及機制

Abstract

恢復心臟功能是細胞治療的最終目標。在動物實驗上細胞治療能使心肌細胞再生，促進心肌血流，並逆轉梗塞後的左心室再塑。然而在臨床上的治療效果卻是分岐、不顯著。這樣的結果顯示我們必須要更了解細胞治療的機制才能發揮應有的效果。本研究有兩部分，分別探討目前用於修復心臟的兩種細胞治療策略：一是幹細胞移植：探討幹細胞移植在陳舊性心肌梗塞的效果及機制，二是幹細胞移動：探討骨髓幹細胞的導引因子包括血管內皮生長因子(vasculoendothelial growth factor)、間質細胞衍生因子(stromal-derived factor 1)及幹細胞因子(stem cell factor)等在心臟復健的角色。 第一部分、幹細胞移植在陳舊性心肌梗塞的效果及機制 這部分研究目的建立陳舊性心肌梗塞的實驗模式並探索細胞治療的效果及機制。幹細胞能否進入心臟取決於兩個間質細胞衍生因子濃度梯度：由骨髓至週邊血液以及由血液至受傷心臟的濃度梯度。然而這些濃度梯度在陳舊性心肌梗塞時降低，造成幹細胞不容易進入心臟。由於骨髓間質細胞會分泌大量的間質細胞衍生因子，同時動物實驗顯示骨髓間質細胞應用於心肌梗塞的治療效果良好。所以本研究探討以骨髓間質細胞為主的細胞治療是否會改變這個濃度梯度。實驗動物為2.5-3公斤的紐西蘭公白兔，以綁扎心臟的冠狀動脈左前降支，綁扎2個月後，隨機分配至食鹽水治療或自體骨髓間質細胞治療。治療方式是以左心室注入，細胞數目為2×106。四週後，與食鹽水治療組比較，細胞組的間質細胞衍生因子的濃度梯度由骨髓至週邊血液及由血液至心臟皆上升。同時增加心肌組織內的幹細胞表面抗原CD34、CD117及STRO-1等陽性細胞數目。心肌組織的微血管密度增加，心臟的收縮及舒張功能改善；心肌梗塞的範圍也減少。這部分研究成果支持「以骨髓間質細胞為主的細胞治療能夠改變間質細胞衍生因子的濃度梯度使之趨向於心臟」。這個效果使得幹細胞更容易進入心臟，協助改善心臟功能。 第二部分、骨髓幹細胞的導引因子在心臟復健的角色 臨床研究部分則是探討心臟復健與幹細胞移動的相關性是否存在。過去研究發現運動訓練能促進骨髓幹細胞由骨髓移動至週邊血液。但以運動訓練為主的心臟復健與幹細胞移動至心臟進行修復的研究仍屬有限。心臟復健被證實可以增加心肌灌流儲備量，而心肌灌流儲備量的增加代表心肌組織內的血管新生，它是一種心臟受傷後修復的表現。所以我們研究心臟復健與幹細胞由骨髓移動到受傷心臟的導引因子如間質細胞衍生因子、血管內皮生長因子及幹細胞因子等關係。有鑒於以往的研究無法將梗塞區域清楚的劃分出來進行分析，所以我們利用心臟核磁共振掃描能夠區分出正常心肌及梗塞心肌的優點，分別探討心臟復健對不同位置的心肌灌流儲備量的影響，再進一步探討梗塞區的心肌灌流改善與幹細胞導引因子的關係。這項研究邀請到39位心肌梗塞病患，經由隨機分配後，其中20位分配至運動復健組，接受長達3個月、每週3次、每次30分鐘的腳踏車或跑步機的運動方式，每次運動量為最大攝氧量的55-70%。其餘19位病患則是保持原來的生活方式。另外有19位健康受試者為健康對照組。與健康對照組比較起來，心肌梗塞患者的心肌灌流儲備量無論在正常心肌或梗塞心肌皆下降，而血漿中的間質細胞衍生因子及血管內皮生長因子濃度則顯著上升。其中只有間質細胞衍生因子與心肌灌流儲備量有顯著相關性，特別是在梗塞心肌內的灌流儲備量(r=-0.62, P<0.001)。經過3個月後，運動復健組的最大攝氧量增加15% (P<0.01)，到達健康對照組的程度。心肌灌流儲備量分別在正常心肌增加30% (P<0.01)，在梗塞心肌增加25% (P<0.05)，兩者皆到達健康對照組的程度。間質細胞衍生因子濃度則下降至健康對照組的程度。最大攝氧量的改變與正常心肌的灌流儲備量改變呈正相關(r=0.55, P<0.001)。間質細胞衍生因子濃度的改變與梗塞心肌灌流儲備量呈負相關(r=0.50, P=0.001)。在沒有運動復健的梗塞患者，運動能力及心肌灌流儲備量沒有改變。所以，心臟復健可以增加正常心肌及梗塞心肌的灌流儲備量，同時增加病患的運動能力。在心肌灌流儲備量上升至正常水準後，導引幹細胞自骨髓移動到受傷心臟的因子如間質細胞衍生因子則下降至正常濃度。 結論及臨床應用 本研究顯示間質細胞衍生因子在細胞治療扮演重要的角色。臨床上幹細胞移植應用在缺血性心臟病患時，若能選用像骨髓間質細胞這種能夠釋放大量間質細胞衍生因子的幹細胞，將可增加心臟修復的療效。此外，本研究不但證實以運動訓練為主的心臟復健可以增加缺血性心臟病患整個心臟的心肌灌流儲備量，也證實梗塞區域的心肌灌流儲備量增加與間質細胞衍生因子，也就是幹細胞自骨髓移動到受傷心臟的導引因子有關。同時心臟復健前血漿中間質細胞衍生因子濃度可能可以做為預測心臟復健成效的因子，值得將來在大規模研究中去證實。

The goal of cell therapy in the ischemic heart disease is to optimize ventricular remodeling and regenerate myocardial structures. However, the treatment effect in humans is modest, in contrast to that in animals, where the effect is marked and significant. Thus, it is necessary to know the mechanism underlying successful cell therapy to resolve the efficacy discrepancy between species. There are two strategies in the current cell therapy: one is stem cell transplantation and the other is stem cell mobilization. It is known stem cell recruitment to heart is determined by a concentration gradient of stromal-derived factor 1 (SDF-1) from bone marrow to peripheral blood and from blood to injured myocardium. However, this gradient is decreased in chronic myocardial infarction (MI). Importantly, bone marrow stromal cells (BMSCs) are cells that can secrete high concentrations of SDF-1. Therefore, in the research of stem cell transplantation, using rabbits as an experimental model of chronic myocardial infarction (MI), we determined whether autologous BMSC transplantation could recruit more stem cells to the heart in order to improve ventricular remodeling, and we explored the changes in SDF-1 levels in the bone marrow, peripheral blood, and myocardium before and after cell therapy. MI was induced in male New Zealand White rabbits (2.5-3kg) by ligation of the left anterior descending coronary artery. Two months later, the rabbits were randomized to either a saline or BMSC group, where the latter received an injection of 2x106 autologous BMSCs into the left ventricular cavity. Four weeks after therapy, the SDF-1 gradients from marrow to blood and that from blood to myocardium increased in the BMSC-treated rabbits compared with saline-treated rabbits. This was accompanied by an increase in cells positive for CD34, CD117, and STRO-1 in myocardium, resulting in more capillary density, better cardiac function, and a decrease in infarct size.We concluded that generation of a SDF-1 gradient toward the heart is a novel effect of BMSC-based cell therapy. This effect facilitates stem cell recruitment to remodeled myocardium and supports improvement in cardiac function. Regarding stem cell mobilization, we study the role of stem cell mobilization in cardiac rehabilization. Cardiac rehabilitation is believed to increase myocardial perfusion reserve (MPR), but this has not been adequately studied because of poor delineation of infarcted myocardium in the previous studies. We determined the effect of cardiac rehabilitation on MPR in the remote and infarcted myocardium with contrast-enhanced magnetic resonance imaging. We then investigated whether cardiac rehabilitation could influence plasma levels of angiogenic cytokines and their correlation with myocardial blood flow (MBF). Thirty-nine postinfarction patients were recruited for this study and randomly assigned to a training group (n = 20) or a nontraining group (n = 19). Those in the training group participated in a 3-month rehabilitation training program at an exercise intensity of 55% to 70% of peak oxygen uptake (VO2), while those in the nontraining group continued their usual lifestyle. Nineteen age-, weight-, and height-matched subjects without cardiovascular risk factors were selected as healthy controls. In the postinfarction patients, a MPR reduction was seen not only in the infarcted myocardium, but also in the remote myocardium. In the training group, exercise capacity increased by 15% (P<0.01) up to the same level as in healthy controls. The post-training MPR was also increased in both the remote (+30%, P<0.01) and infarcted myocardium (+25%, P<0.05) and reached the same level as in healthy controls. The change in exercise capacity correlated with the change in MPR in the remote myocardium (r=0.55, P<0.001 for peak VO2). In the nontraining group, exercise capacity and MPR were unchanged. In conclusion, cardiac rehabilitation improves MPR in both the infarcted and remote myocardium, with a parallel increase in exercise capacity. In addition, postinfarction patients had a higher plasma levels of vasculoendothelial growth factor (VEGF) and SDF-1. Only SDF-1 was inversely associated with stress MBF in both remote and infarcted myocardium (r=0.62, p,0.001). After 3 months, the training group’s stress MBF had increased by 33% in the remote (p<0.001) and 28% in infarcted myocardium (p=0.02), while VEGF decreased by 9% (p=0.01), and SDF-1decreased by 11% (p=0.02). The change in SDF-1 was inversely correlated with the change in stress MBF in both remote (r=0.40, p=0.01) and infarcted myocardium (r=0.50, p=0.001). In the non-training group, MBF and cytokines were unchanged. In conclusions, the present doctoral thesis combined basic research and clinical studies to demonstrate how angiogenic cytokines, especially SDF-1, are involved in the mechanism and efficacy of cell therapy. We first showed the generation of a SDF-1 gradient toward the heart is a novel effect of BMSC-based cell therapy. This effect facilitates stem cell recruitment to remodeled myocardium and supports improvement in cardiac function. Then we performed a prospective and randomized clinical trial showing (1) the possibility of functional recovery of resistant vessels in the infarcted myocardium after cardiac rehabilitation and (2) an inverse relation between plasma SDF-1 and myocardial perfusion, suggesting a feedback regulation of SDF-1 due to increased blood supply to the myocardium after cardiac rehabilitation. This result also indicates that a large prospective study is needed to determine whether including serial measurements of SDF-1 during follow-up improves the ability to detect myocardial hypoperfusion and thereby allows early cardiac rehabilitation.