优化c-kit+间充质干细胞移植和生长因子缓释修复梗死心肌的外源性和内源性机制

Recent studies suggested that cardiac lymphatic edema occurs after myocardium infarction, and production of stem cell factor (SCF) is reduced. The level of growth differentiation factor 11 (GDF11) in old patients is decreased. Therefore, stem cell transplantation is faced with severe influence of local microenvironment. Basing on our recent report that there is a population of c-kit+ cells with potential of differentiation towards myocardiocytes in bone morrow-derived mesenchymal stem cells (MSCs), this program is designed to improve the microenvironment by optimizing of c-kit+ MSC transplantation for promoting repair of the infarcted myocardium. This study investigates potential of c-kit+ MSC differentiation and mechanisms of their survival and differentiation. The functional self-assembling peptide adhering the cells and binding SCF or GDF11 is synthesized. After transplantation, differentiation of the engrafted cells and the endogenous c-kit+ cells and division and proliferation of the myocardiocytes are traced. Myocardial regeneration, angiogenesis and lymphangiogenesis are evaluated. The functional cardiac patch is prepared with 3D biological printing and techniques of organoid culture. After transplantation on the epicardium, migration and differentiation of epicardium-derived cells (EPDCs) are traced. The significance of promoting repair of the myocardium with stem cell transplantation by activating the endogenous repair potential of the epicardium is evaluated. This study will provide new strategies and scientific evidences for treatment of myocardial infarction with stem cell transplantation.

近来发现，心肌梗死后心脏发生淋巴水肿和SCF产生减少，老年患者的GDF11水平降低，故干细胞移植面临不良微环境的严重影响。本项目基于我们最近从骨髓MSCs中分离出具有向心肌分化潜能的c-kit+细胞，拟通过优化c-kit+ MSCs移植改善微环境以促进梗死心肌修复。本研究深入探讨c-kit+ MSCs的分化潜能和存活、分化机制。研制黏附c-kit+ MSCs并缓释SCF或GDF11的功能化自组装肽，移植后示踪移植细胞和追踪内源性c-kit+细胞的分化和心肌细胞分裂增殖，评价心肌再生、血管和淋巴管新生效果。采用3D生物打印和类器官组织培养技术构建功能化心脏补片，心外膜表面移植后追踪心外膜EPDCs的迁移和分化，探讨通过激活心外膜内源性修复潜能显著提高干细胞移植修复梗死心肌的价值。本研究将为临床开展干细胞移植治疗心肌梗死提供新策略和科学依据。

本项目按照原计划要求，圆满完成了各项计划研究内容，达到了预期指标。目前干细胞移植面临两大挑战，一是选择理想种子细胞，二是优化干细胞移植微环境。本项目完成计划概要：①成功地分离和分选了c-kit+MSCs及其亚群，对不同亚群c-kit+MSCs的表型特征和差异表达基因作了综合鉴定。评价了c-kit+MSCs及其亚型细胞的分化潜能，确立了c-kit+VEGFR-2+MSCs作为治疗心血管疾病理想种子细胞的可行性。②合成了黏附干细胞并缓释SCF、GDF11或VEGF-C以及药物Tβ4和维甲酸的功能化自组装肽，综合评价了功能化自组装肽的免疫原性和组织相容性。研制了胶原蛋白三维纳米纤维膜，制备了携带干细胞和药物的功能化心脏补片。这为通过功能化自组装肽优化干细胞移植修复梗死心肌奠定了可靠基础。③明确了SCF和GDF11促进c-kit+MSCs和EPCs存活、分化和抗衰老的自噬机制，深入探讨了SCF/c-kit和GDF11/ALK-4和ALK-5信号途径在c-kit+MSCs和EPCs分化方面的分子机制。④利用大鼠心肌梗死模型在体评价了功能化自组装肽优化干细胞移植和缓释生长因子对于促进梗死心肌修复的效果，明确了优化干细胞移植和缓释生长因子在外源性和内源性修复梗死心肌方面的作用机制。⑤在转基因鼠心肌梗死模型，心肌内注射缓释药物的功能化自组装肽或心外膜表面移植携带干细胞和药物的功能化心脏补片，通过遗传追踪心外膜细胞的激活、迁移和分化，综合评价了优化干细胞移植和药物缓释对心肌再生、血管新生、淋巴管新生和心功能改善的作用，明确了通过激活内源性心肌再生机制显著提高梗死心肌修复效果的可行性。⑥鉴于维甲酸在心脏发育中的重要作用，而成年心脏中的维甲酸含量明显降低，进一步探索了药物缓释通过激活成年心肌细胞分裂增殖促进内源性心肌修复的可行性。这有助于推动优化干细胞移植治疗缺血性疾病的临床医学转化。本项目研究成果已发表SCI收录论文6篇，完成并投稿SCI论文2篇。发表中文核心期刊论文1篇。国际学术会议交流论文6篇，国内学术会议交流论文1篇。在国内学术会议作大会特邀报告1次。邀请海外著名专家来华访问交流与科研合作4次。