可注射Ppy-Chi-hBMSCs-CMs类心肌组织通过抑制心室重构促进心电同步化修复大鼠心梗的实验研究

The massive death of cardiomyocytes (CMs) and the destruction of electrical conduction pathways are two important pathophysiological processes of myocardial infarction (MI). Studies have shown that human bone marrow mesenchymal stromal cells (hBMSCs) transplantation repairs damaged heart tissue. On the other hand, we also found that injection of conductive material restored the electrical impulse propagation through the scar tissue and re-synchronized cardiac contraction. Our group further showed that injection of bioconductive material polypyrrole-Chitosan (Ppy-Chi) accelerates the electrical signal transmission through the myocardial infarct in rat model, but the specific mechanism is still unknown. This study is designated to construct an injectable myocardial-like tissue Ppy-Chi-hBMSCs- CMs and apply it to the rat MI model. Our previous studies confirmed that Ppy-Chi repairs damaged heart by reducing scar tissue resistance, promoting angiogenesis and improving the survival of hBMSCs and CMs. In the present proposal, we hypothesized that Ppy-Chi improves cardiac function by promoting electrical resynchronization and preventing progressive ventricular remodeling post-MI in rats. Electrocardiogram will be used to assess resynchronization of electrical conduction. Echocardiograph will be used to evaluate cardiac function. Bioluminescence imaging, positron emission computed tomography imaging will be used to assess a variety of parameters such as hBMSCs -CMs survival, tissue metabolism and collagen content. To prove the scientific hypothesis: Ppy-Chi improves cardiac function by promoting electrocardiographic resynchronization and inhibiting ventricular remodeling post myocardial infarction in rat.

心肌细胞大量死亡和电传导通路破坏是MI发生的重要病理生理过程。研究证实，hBMSCs移植可以修复受损的心脏组织；导电材料植入可以重构心脏电传导通路；二者单独应用效果不佳。本课题组前期发现，注射导电复合材料Ppy-Chi可以加快大鼠MI电信号传导，但具体机制不明。本研究拟构建可注射 Ppy-Chi-hBMSCs-CMs类心肌组织并应用于大鼠MI模型中，从不同角度证实Ppy-Chi通过降低心脏组织电阻和促进血管生成并提高hBMSCs-CMs存活的途径修复受损心脏组织。利用TDR、DCG、US等技术，寻找Ppy-Chi促进心电传导再同步化证据；利用BLI、小动物PET-CT成像等监测手段，从移植后hBMSCs-CMs存活、心脏组织代谢定量等层面阐明Ppy-Chi降低心脏胶原含量的原因。以证明科学假说：Ppy-Chi通过促进心梗后心电再同步化和抑制心室重构的机制来改善大鼠心脏功能。

目的：探讨可注射导电水凝胶聚吡咯-壳聚糖（Ppy-Chi)联合人子宫内膜间充质干细胞的心肌样细胞（hEMSCs-CMs）治疗大鼠慢性左心室心梗的有效性研究。.方法：以六水三氯化铁为氧化还原剂，戊二醛为交联剂，利用一步氧化还原法构建出Ppy-Chi导电复合水凝胶。在体外，扫描电镜观察其表面颗粒度及多孔结构；循环伏安法检测其导电性能；通过共培养EMSCs,研究其生物相容性；通过体外凝集时间及其质量丢失情况，研究其凝集和降解特性。利用复合酶解法分离EMSCs,采用贴壁培养并传代。应用流式细胞术检测其表面标志物；通过诱导分化实验检测其多向（成脂、成骨、成软骨和成心肌）分化能力。在体内，结扎大鼠心脏左前降支来制备心梗模型，利用心电图、PET-CT来评估模型是否制备成功。造模成功4周后，动物随机分4组（Ppy-Chi-EMSCs-CMs、Ppy-Chi、EMSCs-CMs、对照组），分别将上述材料移植于心梗及周边区，用超声来评估心功能的改善情况。.结果：在本研究中，成功地构建了Ppy-Chi导电水凝胶。与对照组Chi水凝胶相比，Ppy-Chi复合材料具有多孔结构且表面粗糙呈颗粒状，具有良好的导电性。两种水凝胶均支持细胞的生长和增殖，具有良好的生物相容性。以上说明新构建的复合材料是较为理想的细胞载体。Ppy-Chi水凝胶比Chi水凝胶更稳定，更利于其在体内长期发挥作用。通过复合酶消法成功分离hEMSCs并进行传代培养。hEMSCs呈贴壁漩涡状生长，流式细胞术显示P3代hEMSCs高表达CD44、CD73、CD90、CD105（>95%).而CD11b与HLA-DR等呈低表达（<5%)，说明本研究所使用的干细胞纯度较高。并且通过成脂、成骨、成软骨和成心肌的分化检测，均证明该种细胞具有良好的分化潜能。体内实验部分，本研究成功地制备了大鼠心梗模型，心电图显出病理性Q波。将Ppy-Chi-EMSCs-CMs类心肌组织用于大鼠心梗治疗，超声心动图结果显示射血分数和缩短分数均有改善；18F-FDG Micro PET/CT初步探索发现能直接观察到心梗区域。.结论：Ppy-Chi复合导电水凝胶联合EMSCs-CMs可以增强心梗后心功能恢复，改善预后，为缺血性心肌病提供新的治疗策略。