强化阿托伐他汀促进心肌SCF分泌并趋化c-kit+心脏干细胞靶向归巢修复梗死心肌及其机制研究

The premise of this study is based on our previous breakthrough discovery that "statins optimized the efficacy of bone marrow stem cell transplantation after myocardial infarction",as well as "intensive statins therapy bring greater clinical benefit after myocardial infarction",we propose that " intensive atorvastatin (Ator) promote myocardial paracrine of SCF, and promote prolification and self-renewal of c-kit+ cardiac stem cells to repair infarcted myocardium". In vitro, experiments were designed to observe SCF level and chemotactic activity of c-kit+ cardiac stem cells on different doses of Ator in hypoxia/serum-free culture conditions, and clarify the signaling pathway. In vivo, we establish rat acute myocardial infarction model, and randomize to receive low-dose Ator, low-dose Ator + MSCs transplantation, intensive-dose Ator, intensive-dose Ator + MSCs transplantation, as well as intensive-Ator + signaling inhibitor. One and four weeks after MI, the peripheral and myocardial SCF level, target homing of c-kit+ stem cells, neonatal cardiomyocytes and capillaries, and cardiac structure and function are evaluated. It's expected to verify the above hypothesis, and to elucidate its mechanism. Highlight the originality and novel design of this study, it will illuminate new mechanisms of myocardial regeneration and bring important clinical value in the future.

本研究基于前期"他汀优化骨髓干细胞（MSCs）移植疗效"的突破性发现以及"心肌梗死后强化他汀治疗带来更大临床获益"的前提，提出"强化阿托伐他汀（Ator）促进梗死后心肌旁分泌SCF，并动员c-kit+心脏干细胞靶向归巢修复梗死心肌"的假说。拟通过体外实验，观察不同剂量Ator对心肌细胞在缺氧无血清培养条件下分泌SCF的影响，以及对c-kit+心肌干细胞的促增殖作用，并阐明相关信号通路。同时建立大鼠急性心梗模型，随机接受低剂量Ator、低剂量Ator+MSCs移植、强化剂量Ator、强化剂量Ator+MSCs移植、以及强化剂量Ator+信号通路抑制剂。于梗死后1周及4周检测外周及心肌SCF水平、心肌内靶向归巢的c-kit+干细胞数量、新生心肌和毛细血管数量以及心脏结构功能。可望验证上述假说，并阐明其机制。本研究原创性突出，设计新颖，深入挖掘心肌再生治疗的新机制，具有重要的临床应用价值。

基质细胞衍生因子1(stromal cell-derived factor-1，SDF-1)与其特异性趋化因子受体4(CXC chemokine receptor 4，CXCR4)构成的SDF-1/CXCR4轴在细胞迁移的过程中有重要作用。既往研究表明，他汀不但能改善梗死微环境促进间充质干细胞(mesenchymal stem cells, MSCs)存活，而且能增强MSCs的自身抗凋亡能力。然而，他汀能否促进心肌干细胞（cardiac stem cells, CSCs)的迁移和归巢能力尚无相关研究。因此，我们进行此项研究，拟明确阿托伐他汀(atorvastatin，ATV)预处理能否增加CSCs表面CXCR4的表达，并提高CSCs的归巢能力；探讨口服强化ATV联合移植ATV预处理的MSCs (ATV-MSCs) 是否可以进一步改善心梗后心功能，并明确其作用是否是通过SDF-1/CXCR4轴发挥的。体外实验部分：分离并培养C57小鼠CSCs，分为正常对照组、ATV不同浓度梯度组、ATV不同时间梯度组；机制探讨部分设CSCs组、最适ATV预处理组、CSCs+CXCR4中和抗体组、ATV-MSCs+CXCR4中和抗体组。实验表明，与正常对照组相比，ATV剂量依赖性的增加细胞表面CXCR4表达，以1μM ATV处理组最为明显；与正常对照组相比，ATV处理组CXCR4的表达呈一定的时间依赖性，其中ATV处理12小时CXCR4的表达至峰值。Transwell迁移结果显示，ATV预处理组CSCs向SDF-1迁移的数量明显增加，加入CXCR4的中和抗体后CSCs的迁移能力被抑制。体内实验将SD大鼠于AMI后1天、1周和2周测定梗死周边心肌组织SDF-1的动态变化。各组急性心梗模型后1周经颈静脉注射CM-Dil标记的细胞 (2×l06细胞/只)。与AMI组相比，口服强化ATV可显著提高SDF-1的表达，在1周时达高峰，选择1周作为MSCs移植时间点。与MSCs组相比，ATV-MSCs或ATV+MSCs组能够显著提高左室功能，心肌纤维化面积显著减小，炎细胞浸润明显减轻；ATV+ATV-MSCs组与ATV-MSCs组相比各指标均有显著改善。上述指标在给予AMD3100后均受到显著抑制。移植MSCs组的心肌组织中内源性c-Kit+干细胞数显著增多，ATV+ATV-MSCs组进一步提高了c