3D类器官心脏肥大模型的建立及在心血管病治疗中药作用机制解析中的应用

Establishment of 3D organoids model of cardiac hypertrophy and its application in the mechanistic analysis of cardiovascular traditional Chinese medicine

与传统二维(2D)单层培养模式相比,三维(3D)类器官培养能更好地模拟器官组织的生理及病理状态。本研究利用1~3天新生大鼠心脏成纤维细胞(CFs)、心肌细胞(CMs)和内皮细胞(ECs)构建了3D心脏类器官体。动物实验方案经天津中医药大学实验动物福利与伦理委员会审查,符合相关规范。通过观察类心脏直径和搏动情况确定了最佳接种细胞数和培养时间。通过荧光染色对其层次结构和类心脏功能进行评价,发现细胞数为1×10^(4)构建的类心脏微球不仅培养34天后仍可自发性搏动且保持特征细胞层次结构。基于此类心脏微球用苯肾上腺素(PE)为诱导剂构建了心脏肥大模型,并通过线粒体质量、细胞内Ca^(2+)浓度、线粒体膜电位等指标进行评价。为进一步验证所建立模型可用于防治心肌肥大药物的筛选,本研究选用冠心宁注射液(GXNI)进行评价。结果表明GXNI显著逆转了PE导致的心脏微球面积和直径变大,以及线粒体质量、细胞内Ca^(2+)浓度的增加和线粒体膜电位的降低,并减弱心钠肽(ANP)、脑钠肽(BNP)和β心肌肌球蛋白重链(β-MHC)的表达上调。本研究成功建立了诱导心脏肥大导致心脏重塑的3D类心脏体外模型,在此体系中,心脏球状体具有类心脏形态及细胞外基质成分,且表现出自发及节律的收缩舒张功能,提示类心脏微球有潜力作为研究心脏肥大病理机制及筛选相关药物的有效模型。

Compared with the traditional two-dimensional(2D)monolayer culture,three-dimensional(3D)organoid can better simulate the physiological and pathological microenvironment of organs and tissues.In this study,3D cardiac organoids were constructed using cardiac fibroblasts(CFs),cardiac myocytes(CMs)and endothelial cells(ECs)isolated from hearts of 1-3-day Sprague-Dawley(SD)neonatal rats.The experimental scheme was approved by the Experimental Animal Welfare and Ethics Committee of Tianjin University of Traditional Chinese Medicine and met the standards of experimental animal welfare and ethics.Optimal seeding cell density and culture time were determined by observing the sphere diameter and pulsation.The hierarchical structure and cardiac-like function were evaluated by fluorescence staining.The results showed that the cardiaclike microspheres constructed with cell number of 1×10^(4)still beated spontaneously even after 34 days in culture,and maintained characteristic cellular hierarchical structure.Then,based on these cardiac microspheres,a phenylephrine(PE)-induced cardiac hypertrophy model was established and evaluated by mitochondrial mass,intracellular Ca^(2+)concentration and mitochondrial membrane potential.Guanxinning Injection(GXNI)was tested to verify that the established model can be used for myocardial hypertrophy drug screen.The results showed that GXNI significantly reversed the enlargement of cardiac microsphere area and diameter,the increase of mitochondrial mass,intracellular Ca^(2+)concentration and the decrease of mitochondrial membrane potential caused by PE,and reduced upregulation of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain(β-MHC).In conclusion,this study successfully established a 3D in vitro model of cardiac remodeling induced by cardiac hypertrophy.In this new system,cardiac microspheres not only have cardiac-like morphology and extracellular matrix components,but also exhibit spontaneous and rhythmic systolic and diastolic function.Therefore,the cardiac microsphere is an effective model to investigate the pathological mechanism of cardiac hypertrophy and screen related drugs.