基于iPS细胞构建组织工程心肌薄片促进心梗后心肌修复的实验研究

Heart failure following myocardial infarction(MI) is the most common cause of coronary disease deaths. Modeling cardiovascular diseases with hiPSC-CM in vitro and stem cell therapy after myocardial infarction are the focus of cardiovascular tissue engineering research in recent years. Our previous have confirmed the extracellular environment played a critical role for the physiology of hiPSC-CM in the process of hiPSC-CM development and maturation in vitro. Therefore, we speculate that left ventricular extracellular matrix remodeling after myocardial infarction changes the characterization of extracellular matrix and these changes ultimately influence resident hiPSC-CM and limit the potential of therapeutic strategies to regenerate cardiac tissue. This project aims to develop the engineered myocardium slice which consists of hiPSC-CM seeded onto decellularized porcine myocardium slice that retains important biochemical, structural, mechanical and functional aspects of native myocardium, based on the tissue engineering principles, direct differentiation of hiPSC and 3D culture technology. Meanwhile, the engineered myocardium slice can be developed to an easy preparation and regulatory tissue-level model for disease research and used to illustrate the physiological and pathophysiological roles of myocardial extracellular matrix remodeling after myocardial infarction on adhesion, growth and function development of hiPSC-CM. Finally, we will utilize the animal model of MI to investigate the treatment advantages of engineered myocardium slice encapsulated with hiPSC-CM on myocardial regeneration and heart function improvement after infarct myocardium. Findings of this study will provide new ideas and theoretical basis to improve the therapeutic effect of stem cell therapy after myocardial infarction.

心肌梗死后心功能衰退是冠心病最常见的死亡原因。制备组织工程化心肌、利用诱导多能干细胞-心肌细胞(hiPSC-CM)建立疾病模型及心梗后干细胞治疗是近年来心血管领域研究的热点。课题组前期研究发现细胞外环境在hiPSC-CM发育过程中发挥关键作用；推测心梗后梗死部位心肌细胞外基质重构影响干细胞移植心肌修复效果。本项目基本思路如下：1.以hiPSC定向诱导分化及天然组织去细胞技术为基础，实现心肌组织去细胞后种植hiPSC-CM再细胞化，制备组织工程心肌薄片，建立具备天然心肌组织细胞外基质结构的体外三维细胞培养体系；2.以此组织工程心肌薄片作为体外研究模型，探究心梗部位重构的心肌细胞外基质对干细胞黏附、存活以及功能发育的影响；3.建立心梗动物模型，比较组织工程心肌薄片移植与传统干细胞移植治疗心肌梗死的疗效，明确其促进心肌修复、改善心功能的作用，为提高心梗后干细胞疗法的治疗效果提供新思路。

干细胞移植促进心梗后心肌修复再生是目前具有较高临床应用前景的心肌梗死后治疗方式之一，研究显示将hiPSC诱导分化成为心肌细胞(Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, hiPSC-CMs)移植于心梗部位后，hiPSC-CMs能一定程度整合进入宿主心肌组织，并与宿主心肌细胞产生电耦合，促进心肌组织修复再生；但hiPSC-CMs移植后定植率及存活率较低，宿主心功能改善不明显，治疗效果受到限制，因此寻找干细胞移植的最佳植入方式及高移植细胞定植率及存活率是亟待解决的关键问题，对于提高干细胞移植治疗效果具有重要意义。本项目以hiPSC定向诱导分化及天然组织去细胞技术为基础，通过心肌组织去细胞后种植hiPSC-CMs实现再细胞化，成功制备了具有独立自主知识产权的载hiPSC-CMs组织工程心肌薄片，通过检测其细胞学、组织学、电生理学及生物力学特征，证实了其生物学活性；同时深入探究了hiPSC-CMs电生理学特征，系统描绘了多种复极钾通道，如快速和慢速延迟整流电压门控K+电流（IKr和IKs）、内向整流K+电流（IK1）、超快速延迟K+电流（IKur）等在hiPSC-CMs动作电位复极中的作用；进一步以此组织工程心肌薄片模型，体外实验证实了心梗后心肌细胞外基质重构以及肾上腺能受体激活对hiPSC-CMs移植后生物学及电生理学功能的影响；最后通过前降支结扎法建立巴马猪心梗模型，通过超声及影像学检测比较了载hiPSC-CMs组织工程心肌薄片移植与单纯hiPSC-CMs移植改善心梗动物模型心功能及左室形态的作用，证实了载hiPSC-CMs组织工程心肌薄片移植促进心梗后心肌修复、改善心功能的优势，为提高心梗后干细胞疗法的治疗效果提供了新思路及新方法。