功能性新型聚合物微泡在体示踪骨髓间充质干细胞治疗急性心肌梗死的研究

Bone-marrow derived mesenchymal stem cells (MSCs) are a promising treatment for heart diseases resulting from myocardial infarction. Cell-based therapies have generated enthusiasm as a new approach to repair or regenerate tissue caused by cardiomyocyte death. Early clinical trials of intracoronary infusion of autologous unfractionated bone marrow have suggested therapeutic benefit. There is pressing need for in vivo methods to track administered stem cells in human populations in order to optimize treatment protocols. Current methods for imaging stem cells in vivo have major limitations. Safety issues that potentially limit these technologies include the requirement for radiolabeling or genetically modifying the stem cells, radiation exposure to the patient, and toxicity of iron particles. Contrast-enhanced ultrasound imaging of stem cells may have unique advantages for in vivo real time monitoring of cell therapy in humans. We propose to use ultrasound contrast agents to pre-label therapeutic MSCs, rendering them visible during diagnostic medical ultrasound imaging. Ultrasound contrast agents are gas-filled microbubbles encapsulated by a biocompatible shell, which we can synthesize with precision to a specific size. We will synthesize a unique microbubble with a biodegradable polymer shell, which will degenerate as the hydrogen ion concentration in MSC changed. In the current proposal, we will render MSCs, which would otherwise be ultrasonically‘invisible’ultrasonically detectable by labeling them with gas-filled microbubbles. We will systematically test rationally designed formulations of polymer microbubbles with the goal of developing a new ultrasoundbased imaging strategy for serially tracking therapeutically delivered MSCs. We will confirm that polymer microbubbles retain acoustic activity after internalization by MSCs and that MSCs become ultrasonically visible in vitro upon internalization of microbubbles and microbubble labeling of MSCs does not change major cell functions of MSCs. And we will determine whether microbubble-labeled MSCs can be ultrasonically imaged in vivo after intramuscular injection of microbubble-MSC complexes into mouse thigh muscle or infarcted myocardium. These studies will lead to a search for new treatment strategies to repair the heart, including delivery of reparative cells to injured tissue

骨髓间充质干细胞（Mesenchymal Stem Cells, MSCs）移植治疗急性心肌梗死可明显缩小梗死区面积，改善心室功能，已在临床得到开展。但现有的干细胞在体示踪成像技术存在分辨率低、易出现假阳性、需要对干细胞进行放射标记或基因修饰等方面的缺陷，在临床应用中受到限制，而超声造影气体微泡具备在体示踪干细胞的理想条件。本项目拟制备一种新型功能性聚合物微泡，较脂质微泡稳定，具有pH值敏感性，用于“标记”骨髓MSCs，在细胞内长期“存活”并随着细胞的凋亡而消失，使干细胞达到“可视”效果。我们将在体外实验中优化微泡标记MSCs的条件、检测标记后MSCs细胞活性；在裸鼠后肢肌肉内及心肌梗死模型的体内实验中探索超声成像条件，并在MSCs移植后的不同时间点应用超声成像技术进行实时监测，旨在实现干细胞在体示踪，为深入了解干细胞在修复受损心肌过程中的机制、完善干细胞治疗方案提供了一种全新手段。

急性心肌梗死（Acute myocardial infarction, AMI）是冠状动脉急性或持续性缺血缺氧所引起的心肌梗死，造成心肌细胞的死亡和心功能的降低，最终可导致心力衰竭。心脏自我修复和心肌再生能力非常有限，故急需寻求一种新的修复受损心肌的治疗措施。有实验证实骨髓间充质干细胞（Mesenchymal Stem Cells, MSCs）可明显缩小心肌梗死区面积，改善心室功能，若能实现干细胞的在体示踪，则可进一步优化治疗方案。超声造影（Contrast-enhanced ultrasound, CEUS）可明显提高图像分辨力，造影剂微泡直径可达到纳米级，具备在体示踪干细胞的基本条件。本研究中，我们制备了一种新型的功能性聚合物微泡，能够在细胞内长期“存活”，且具有pH值敏感性，在细胞死亡后，随着pH值的改变而失去声学特性，然后使用这种微泡“标记”骨髓MSCs，通过超声成像使干细胞达到“可视”效果，从而实现干细胞的在体示踪。在实验中，我们成功向传统的PLGA聚合物体系中引入了pH敏感的聚合物嵌段共聚物PEO-b-P（DEA-stat-TMA），制备了pH敏感的新型聚合物微泡，微泡粒径达到223nm，具备实现干细胞在体示踪的基本条件；在体外条件下，我们成功构建了MSC-MB复合体，并证实了聚合物微泡标记对骨髓MSCs活性不造成影响；随后在自制体外成像模型中，我们确定了超声频率为14MHz、-15dB时MSC-MB复合体成像效果最理想；在此条件下我们实现了MSC-MB复合体的体内成像。最终，我们在小鼠心肌梗死区域注射MSC-MB复合体，并在不同时间点对细胞移植后小鼠的心功能进行评价，证实了MSC-MB复合体对心肌梗死小鼠的心功能具有改善作用，实现了干细胞的“可视”，为临床利用干细胞移植修复受损组织提供了一种全新的监测手段。