人羊膜上皮细胞调控miRNA-1与miRNA-133抗细胞凋亡治疗心肌梗死的机制研究

Myocardial infarction (MI) refers to the myocardial necrosis resulted from serious and persistent ischemia which occurs when coronary artery lesion causes reduction or interruption of blood supply. Cell apoptosis is one of the ways that ischemia causes myocardial cell death. Recent study found that MicroRNA (miRNA)-1 and miRNA-133 specifically highly expressed miRNA in the mature myocardial tissues, which play an important regulatory role in MI and cell apoptosis. We observed in our previous work that in the rat model of acute MI, human amniotic epithelial cells (h-AECs) differentiated into cardiomyocyte-like cells in vivo, reduced the infarct area, and improved cardiac function. This project intends to observe the influences of h-AECs transplantation on myocardial cell apoptosis and on expressions of miRNA-1 and miRNA-133 through the acute MI model in rats in vivo and in vitro, and to explore the target gene and signal transduction mechanism for h-AECs to inhibit myocardial cell apoptosis, in order to demonstrate the hypothesis that h-AECs inhibit myocardial cell apoptosis and protect myocardium by regulating the expressions of miRNA-1 and miRNA-133, discuss the molecular mechanism of its anti-apoptosis role, and provide a solid theoretical basis for application of h-AECs in stem cell therapy for MI.

心肌梗死是由冠状动脉病变引起血供减少或中断，使心肌严重持久缺血所致的坏死。细胞凋亡是缺血所致心肌细胞死亡的途径之一。近年发现，MicroRNA (miRNA)-1与miRNA-133是在成熟心肌中特异性高表达的miRNA，在心肌梗死及细胞凋亡中发挥着重要的调节作用。我们在前期工作中观察到，在大鼠急性心肌梗死模型下，人羊膜上皮细胞分化为心肌样细胞、减少梗死心肌面积、改善心功能。本项目拟通过建立在体和体外大鼠急性心肌梗死模型，观察人羊膜上皮细胞移植对急性心肌梗死心肌细胞凋亡的影响，人羊膜上皮细胞移植对miRNA-1与miRNA-133表达的影响，人羊膜上皮细胞抑制心肌细胞凋亡的靶点基因、信号转导机制，以论证人羊膜上皮细胞调控miRNA-1与miRNA-133的表达，抑制心肌细胞凋亡，保护心肌的假说，探讨其抗细胞凋亡的细胞分子机制，为人羊膜上皮细胞在心肌梗死干细胞治疗应用上提供坚实的理论依据。

申请人通过前期工作发现人羊膜上皮细胞移植可以再生心肌，减少梗死心肌面积，改善心功能，然而仅有3%左右的植入细胞可以分化为心肌细胞。据此推测，人羊膜上皮细胞发挥对损伤心肌组织的修复及再生作用，并不完全依赖于其移植分化，抗细胞凋亡等旁分泌功能起着更重要的作用。本研究第一阶段，课题组首先通过冠状动脉结扎诱导的大鼠急性心肌梗死模型，观察人羊膜上皮细胞移植前后心肌细胞凋亡的变化。结果显示，人羊膜上皮细胞移植及人羊膜上皮细胞条件培养液可以减轻心肌细胞的凋亡。第二阶段，本课题组在上述工作的基础上，观察人羊膜上皮细胞移植前后miRNA-1与miRNA-133表达的变化。结果显示，人羊膜上皮细胞调控心肌特异性miRNA-1低表达，miRNA-133高表达。第三阶段，通过体外培养大鼠心肌细胞，建立心肌梗死缺血、缺氧模型，给予人羊膜上皮细胞条件培养液、miRNA-1与miRNA-133模拟剂及抑制剂干预缺血、缺氧损伤的心肌细胞后，观察心肌细胞凋亡的变化；在体及体外，观察Bcl-2、Bax表达的变化；t-JAK2、p-JAK2、t-STAT3及p-STAT3蛋白表达的变化。结果显示，人羊膜上皮细胞调控心肌特异性的miRNA-1与miRNA-133的表达，启动JAK/STAT信号通路，控制下游靶基因Bcl-2与Bax的表达，减少心肌细胞凋亡，减轻梗死后心肌缺血损伤。综合上述结果，本研究为人羊膜上皮细胞移植治疗急性心肌梗死的作用机制奠定了坚实的工作基础，使人羊膜上皮细胞在再生医学领域具有重要的应用价值和广泛的应用前景。