腱生蛋白C（Tenascin-C）在心脏干细胞心肌修复中作用的研究

Evidence suggests that cardiac stem cells (CSC) enhance heart function after acute myocardial infarction (AMI) via paracrine effects. The effects on heart function recovery are limited because a rare of population cells could be homing in infracted myocardium. It was showed that tenascin-C was expressed by myocardium in border zone during the acute stage after myocardial infarction and regulated several cellular functions associated with cell migration, proliferation and differentiation. Tenascin-C is also an endogenous activator of Toll-like receptor 4 (TLR-4). However, it is not clear whether tenascin-C regulates CSC by activating TLR-4. In our previous study, we have demonstrated that serum TN-C level increased in AMI patients. In addition, TN-C enhances MSCs migration in vitro. However, it is still unclear if the effects of TN-C on CSC via its TLR-4 after CSC transplantation in AMI therapy. Thus, in present study, CSC is isolated from TLR-4 knockout mice, Tenascin-C knockout mice and wild mice. After cultured with tenascin-C, the effects on CSC migration, proliferation and differentiation are explored in vitro. Furthermore, CSC is implanted into mice post-infarction. The effects of tenascin-C on heart function, CSC migration, proliferation and differentiation are investigated in vivo. It would provide new theoretical basis on stem cell therapy after myocardial infarction.

心脏干细胞（CSC）通过旁分泌作用促进梗死后心肌修复，但CSC归巢至梗死心肌区域数量少，改善心脏功能有限。已证实心肌梗死区“边缘”心肌表达的腱生蛋白C通过其活性配体Toll样受体-4（TLR-4），促进多种细胞均有迁移、增殖及分化表达调节。CSC、心脏血管及内皮均表达TLR-4，我们的前期研究发现心肌梗死后患者血清Tenascin-C升高且Tenascin-C可以促进体外间充质干细胞迁移，但Tenascin-C是否作用于CSC及其表达的TLR4，并对CSC移植产生影响，目前尚不清楚。本研究拟获取TLR4-/、Tenascin-C-/-及野生型小鼠CSC；观察Tenascin-C对CSC的增殖、分化与迁移的影响；在体观察CSC移植到上述心肌梗死小鼠体内后，CSC归巢、增殖、分化及心脏功能的变化与Tenascin-C/TLR-4的关系，从而为提高CSC移植治疗心肌梗死疗效提供新的理轮依据。

干细胞移植能改善梗死后心脏功能，仍然面临细胞定植率低、心肌微环境不适合等问题。本课题探究Tenacin-C 对 iPSC 移植治疗心肌梗死改善心肌修复效率的影响及作用和机制。通过构建野生型和TN-C基因敲低小鼠心肌梗死（MI）模型，在体内和体外探究TN-C对MI后心肌细胞焦亡的作用，以及体内体外实验探究TN-C干预诱导多能干细胞（hiPSC）/骨髓间充质干细胞（BMMSC）修复梗死后心肌疗效和机制。已完成相关焦亡信号通路的机制研究，初步探究了TN-C提高iPSC/BMMSC移植修复梗死心肌疗效的作用机制。本研究在分子、细胞和组织水平，用免疫学和分子生物学的方法，证实MI心肌梗死所致的TN-C上调与caspase-1依赖性细胞焦亡之间的直接联系，且这一过程部分由TLR4/NF-kB/NLRP3和IL-18、IL-1β信号通路介导。获取人iPSC/BMMSC移植治疗MI小鼠，通过超声心动图、活体成像和分子生物学的方法，在蛋白、基因水平上，证实hiPSC移植治疗能够改善心肌梗死小鼠的心脏功能，而应用TN-C预处理的hiPSC能够使小鼠的心功能得到进一步改善。其可能的机制为，TN-C促进hiPSC归巢，增加干细胞的定植效率，在hiPSC移植的微环境下，激活Wnt/β-catenin通路，减少胶原沉积和心肌纤维化，促进心肌修复。本课题探究了TN-C在梗死后心肌损伤和修复中的多重功能。通过构建体内体外MI模型、TN-C基因下调以及不同的hiPSC治疗方案阐明了，TN-C在梗死后急性期心肌损伤中的作用，以及外源性补充TN-C能够增强hiPSC修复心肌疗效的机制。