mTOR信号转导通路在间充质干细胞治疗急性心肌梗死中的作用机制及分子影像评价

Mechanism of MSCs transplantation in curing ischemic heart disease has not yet been fully elucidated. This study aims to use the Chinese mini-swine myocardial infarction model to confirm what we proposed, that MSCs transplantation improves cardiac function not only by differentiation and angiogenesis of MSCs, but also improve the energy substrate metabolism of cardiocytes, and then improve cardiac contractility by means of mTOR signal transaction pathway. In this study, we will investigate the effect and mechanism of MSCs transplantation in AMI by means of detecting glucose metabolism in global left ventricular myocardium and regional myocardium, combined with assessment of cardiac function, and also to study the changes of mTOR pathway proteins, glucose metabolism-related enzymes which may participate in the MSCs paracrine process by means of RT-PCR, Western-Blot and fluorescence immunoassay. Moreover, we will in vivo observe the changes of myocardial glucose metabolism, myocardial perfusion and cardiac function after MSCs transplantation in swine with AMI by means of cell biology and molecular imaging methods including positron emission tomography-computer tomography (PET-CT), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI), in the purpose of evaluating the effect and mechanism of MSCs transplantation treating ischemic heart disease..This project aims to use cytobiology, molecular biology and molecular imaging technology, to monitor the effects of stem cell transplantation on ischemic myocardium, and to evaluate the influence of mTOR signal transaction pathway of stem cell on cardiac systolic function, and then to develop the clinical application of stem cell transplanting treatments...

干细胞移植治疗急性心肌梗死的作用机理尚未完全阐明。本项目在前期研究的基础上，利用中华小型猪心肌梗死模型，通过移植间充质干细胞（MSCs），探讨PI3K/Akt/mTOR/4E-BP1信号转导通路在MSCs旁分泌治疗缺血心肌的作用机理。主要研究内容包括：利用细胞生物学及分子生物学的方法，从分子层面探讨mTOR通路对MSCs移植的影响；通过SPECT、PET及MRI等分子影像学方法，在不同mTOR通路状态下动态观察MSCs移植后缺血心肌的血流灌注、葡萄糖代谢及心功能的变化；通过RT-PCR、Western-Blot及荧光免疫技术观察mTOR通路在活化状态下各通路蛋白及葡萄糖代谢酶PFK、GAPDH等基因与其蛋白的表达情况。本项目旨在通过细胞生物学、分子生物学及分子影像学技术，评价mTOR信号转导通路对MSCs移植后心肌细胞代谢及心功能的影响，对于完善临床干细胞移植治疗方案具有很大的应用价值。

急性心肌梗死因其高发病率和死亡率，严重影响人类的预期寿命和生存质量。骨髓间充质干细胞（bone marrow mesenchymal stem cells，BM-MSCs）是急性心肌梗死（acute myocardial infarction，AMI）干细胞移植治疗的理想供体之一，干细胞移植治疗心血管疾病为心肌梗死的治疗带来了新的契机，作用机制仍是争论和研究的焦点。除了多数研究确定的血管生成作用，我们课题组推测可能是MSCs移植后，其旁分泌的细胞因子激活了mTOR信号转导通路，p70s6k作为下游的效应分子促进了心肌葡萄糖的代谢。本研究将细胞生物学与正电子发射计算机断层扫描-计算机断层扫描（PET-CT）、单光子发射计算机断层扫描（SPECT）等分子影像检测手段相结合，并通过MRI检测，重点观察在体动物MSCs移植治疗AMI后心肌血流灌注、心肌葡萄糖代谢及心功能的改变，进而评价MSCs治疗心肌血流灌注及糖代谢的变化，并通过分子生物学手段对可能参与MSCs旁分泌作用的mTOR信号转导通路进行初步的研究，并观察葡萄糖转运体、葡萄糖代谢相关酶的变化，探索MSCs移植治疗的疗效及机制。.在本研究中我们利用心肌灌注SPECT评价了干细胞移植后的心肌血流灌注；利用 PET/CT评价了干细胞移植后心肌葡萄糖代谢的变化；通过MRI评价了干细胞移植后心肌收缩功能的改变；监测干细胞移植后，细胞移植局部心肌组织中糖代谢关键酶及与糖代谢密切相关的细胞因子浓度变化趋势与局部室壁收缩功能改善的关系；研究了干细胞移植后mTOR受体通路的改变。 .本课题前期研究结果显示：经心肌内注射骨髓MSCs治疗AMI，4周后心功能明显改善，心肌血流灌注及心肌葡萄糖代谢提高，推测心功能的改善与血管生成及心肌糖代谢增加相关。注射MSCs的区域心肌细胞葡萄糖转运体、mTOR蛋白及其通路下游分子p70s6k表达显著升高，提示MSCs可能通过旁分泌作用激活该通路，促进心肌葡萄糖代谢及细胞生长，进而增加心肌收缩、促进心功能改善。