阿托伐他汀提高c-kit(+)心肌干细胞治疗急性心肌梗死的疗效及其机制

It is in debate about that adult stem cells trans-differentiate into cardiomyocytes, and it is the most important that the therapeutical effects of adult stem cells is very limited to treat acute myocardial infarcton (AMI), which is a hamper to be translated into clinical settings. However, as a new cell population recently isolated from adult hearts of animals and human beings, cardiac stem cells (CSCs) may be the most appropriate candidate cell for transplantation because of their powerful and definate differentiation potential into cardiomyocytes in vitro and in vivo. Unexpectedly, the theraputic effects of CSCs in AMI animals and huma beings are inconsistent. Since we designed and validated the hypothesis that statins protected the implanted mesenchymal stem cells in the post-infarction cardiac microenvironments in vitro and in vivo previously, in the current project we hypothesize that statins inhibit the apoptosis of CSCs to enhance their survival and efficacy via HGF/c-Met-PI3k-Akt signal pathway. The research contents include: 1)to establish a system of isolation, culture and differentiation of c-kit positive CSCs of rat in vitro, to explore the impacts of Atorvastatin on the apoptosis of CSCs induced by hypoxia/serum-free in vitro, and subsequently to test what role HGF/c-Met-PI3k-Akt signal pathway plays during the above process. (2) to explore whether Atorvastatin enhance the survival and therapeutic effects of CSCs implanted into the post-infarction cardiac microenvironments in rat models of AMI in vivo, and then to test the potential of HGF/c-Met-PI3k-Akt pathway in the protective effects of Atorvastatin in vivo. The present project will provide a new strategy and method to resolve the international problem that transplantation effects of adult stem cells are significantly limited to treat AMI, which is not reported previously and endowed with important scientific and translational value.

干细胞治疗急性心肌梗死（AMI）的疗效低下已成为其转化应用的国际难题，尽管心肌干细胞（CSCs）具有确凿的心肌细胞分化能力，然治疗AMI效果仍存在争议。基于我们前期研究证实他汀通过改良梗死心肌微环境而提高移植骨髓干细胞的存活和疗效，进一步假设：他汀通过HGF/c-Met信号途径抑制CSCs凋亡，提高其移植后的存活和疗效。通过体外建立心肌缺血的细胞模型诱导大鼠c-kit（+）CSCs凋亡，研究阿托伐他汀对CSCs凋亡的影响和最佳浓度，及HGF/c-Met信号系统在其中的作用；通过大鼠AMI模型，验证他汀通过抑制细胞凋亡提高CSCs在梗死心肌中的存活和疗效，进一步应用HGF拮抗剂NK4阐明HGF/c-Met信号途径在他汀保护CSC机制中的作用。为解决成体干细胞移植治疗AMI疗效低下这一突出国际难题提供全新策略和方法，具有重要的科学意义和转化医学价值，国际上未见类似报道。

经本项目的资助，我们通过提取ATV预处理后CSCs分泌的外泌体（CSCATV-Exo）治疗急性心肌梗死（AMI），结果显示CSCATV-Exo可以显著改善AMI大鼠心功能、缩小梗死面积，且该效应与内皮细胞存活和功能改善有关。主要发现如下：（1）通过超速离心法成功提取ATV预处理24h后CSCs分泌的Exo（CSCATV-Exo），经鉴定表达外泌体特异性标记，透射电镜可见呈直径约为100nm的典型杯口样形态。（2）CSCATV-Exo可使低氧和无血清(H/SD)处理的人脐静脉内皮细胞凋亡减少，迁移能力和形成毛细血管能力增强，该效应与ATV预处理后MSCs分泌的外泌体（MSCATV-Exo）类似，且强于未经ATV预处理的CSCs和MSCs产生的Exo（CSC-Exo和MSC-Exo）。（3）通过在体实验，在AMI造模30分钟后心肌内注射CSCATV-Exo，分别于造模前、AMI后3天、7天和28天行超声心动图检查，并于第28天取材行组织学检测，发现CSCATV-Exo注射可显著改善大鼠心梗后心功能、抑制心室重构、减轻心室纤维化、缩小梗死面积，且程度优于CSC-Exo。上述发现证实CSCs可通过分泌Exo产生心肌保护作用，且他汀处理可提高其效率，为AMI后心肌再生修复提供了新的干预方法。