心肌仿生细胞外基质生物补片促进心肌梗死后心肌修复

Stem cells are promising therapeutic agents for myocardial infarction (MI). Destruction of extracellular matrix (ECM) following MI often contributes to poor survival and retention of the grafted stem cells. Our previous study confirmed that nanofibrous patches can promote the survival of the loaded stem cells and myocardial repair after MI. Our group also found that CTRP9 can improve myocardial adverse microenvironment after MI, resulting in enhanced proliferation, migration and survival of transplanted stem cells. However, CTRP9 has a short half-life and is difficult to function continuously in ischemic myocardium. Studies found that peptide analogues could be covalently crosslinked to a collagen scaffold via dendrimers that are rich in amine groups (-NH2), constituting a controlled delivery system. Based on aforementioned preconditions, we propose the following scientific hypothesis: The myocardial biomimetic ECM with sustained release CTRP9 is loaded with multi-layer stem cells to construct biologic patches. The aim of the project is to explore the myocardial repair effect of the patch from three aspects: myocardial structure and microenvironment (biomechanical characteristics of biomimetic ECM and sustained release of CTRP9), the amount of stem cells (multilayer stem cells and pro-survival factor CTRP9) and function of the stem cells (CTRP9 promotes the proliferation, migration and paracrine function of the stem cells).

干细胞是心肌梗死（MI）很有前景的治疗手段，而MI后心肌细胞外基质（ECM）的破坏及伴随不良微环境导致移植干细胞急性流失和死亡，限制了其临床转化。申请人发现纳米纤维补片可促进所承载干细胞的存活和MI后心肌修复（Acta Biomaterialia 2018）。本课题组前期发现CTRP9是维持健康心肌微环境的关键因子，可促进MI后移植干细胞的增殖、迁移和存活（Circulation 2017）。然而CTRP9半衰期短，需全身给药，对缺血心肌的作用有限。有研究发现聚酰胺聚合物可使肽类交联于胶原蛋白实现缓释。我们提出科学假设：可缓释CTRP9的心肌仿生ECM与多层干细胞结合构建生物补片，从三个方面：心肌结构和微环境（仿生ECM的生物力学特征和CTRP9缓释）、干细胞数量（多层干细胞和促存活因子CTRP9）和干细胞功能（CTRP9促进干细胞增殖、迁移和旁分泌功能），探讨补片的心肌修复效果。

背景：纤维连接蛋白(fibronectin，FN)聚合和胶原蛋白过度沉积是心肌损伤后心肌纤维化和心室重塑的关键环节，这一过程受机械力的调节。本课题假设通过具有一定力学特征的FN-COL交联心肌补片抑制心室扩张及其所致的心室壁机械张力，可减轻心梗后FN聚合和胶原蛋白沉积，抑制心肌纤维化和心室重塑。.方法：心肌补片是由Ⅰ型collagen和fibronectin通过自组装交联和水凝胶压缩塑形将制备而成的一种具有一定生物力学性能的致密蛋白膜。流变仪检测补片的弹性（杨氏模量）、粘合性和刚度（组织顺应性），扫描电镜观察其微观结构和细胞相容性。前降支结扎法建立小鼠心梗模型，术中植入心脏补片。超声心动图检测心脏结构和功能。病理组织学染色评估心肌纤维化和心室重塑。和WB（α-SMA、Collagen I、MMP2、TGF-β1、P-Smad3、BNP）评估心肌纤维化和心室重塑。.结果：FN-COL patch相较COL patch力学性能（弹性）相当，但具有更好粘合性（流变仪）。超声心动图表明FN-COL补片可改善心梗后的心脏结构（舒张末容积和收缩末容积降低，室间隔厚度增厚）和功能（EF和SF升高）。组织病理学染色和WB检测发现，相比COL patch，FN-COL patch在移植后可与心外膜进行更紧密的整合。这种紧密的整合保证了补片可以抑制心梗后的心室扩张。FN-COL patch还可促进冠脉重建，抑制FN聚合和MFs激活（α-SMA表达下调）。FN-COL patch抑制胶原蛋白合成和心肌纤维化的关键信号通路（TGF-β1/P-Smad3/MMP2 /Collagen I）和心室重塑（BNP表达）的水平。.结论：外源性FN-COL心肌补片可抑制心梗后心室扩张，抑制内源性FN聚合和胶原蛋白的沉积，减轻心梗后心肌纤维化和心室重塑。组织工程心肌补片作为心肌结构和力学强度的补充，是一种治疗心梗后心衰的有前景的治疗手段。