胰高血糖素样肽-1 对脂肪来源干细胞移植治疗心肌梗死的作用和机制研究

There are great potentials for stem cells-based therapy in treating myocardial infarction. With the low cell retention after transplantation, myocardial microenviroment in infarcted region was rendered as a key target. Moreover, stem cells can improve impaired myocardium via paracrine and differentiation mechanisms. Glucagon-like peptide-1 (GLP-1), as one of endogenous active substances, can exert pleiotropic biological effects by stimulating several signal pathways. Our pre-stage study had also found that cell adhesion capacity was related to oxidative stress. In this project, we first plan to study on the effect of GLP-1 on in vitro oxidative injury to adipose derived stem cells (ADSCs), explore its ability to improve myocardial microenvironment, and clarify GLP-1 molecular mechanism through which stem cells' resistance to damage were enhanced. Then, as the key paracrine proteins and signaling pathways were selected, we focus on the ADSCs' paracrine expression efficiency and differentiation into myocardiocyte by pretreating GLP-1. At the moment using real-time in vivo tracking technique, we try to elucidate GLP-1's intervention in infracted region in terms of cells survival, proliferation and differentiation. After that, through functional and histological assessments, we aim to eventually develop and also validate the effective treatment of myocardial infarction by ADSCs transplantation model based on GLP-1 agent.

干细胞移植治疗心肌梗死具有重大前景。植入细胞存活率低是制约疗效的关键瓶颈，而心梗区域微环境是影响细胞存活的主要因素。干细胞定植入梗死区域后，通过旁分泌和细胞分化发挥修复作用。胰高血糖素样肽-1（GLP-1）是一种内源性血糖调节激素，可激活多种信号通路发挥多效性作用。本项目在前期发现细胞粘附能力与氧化应激相关的基础上，拟对GLP-1减轻脂肪来源干细胞（ADSCs）体外氧化应激损伤的作用进行研究，探讨其改善移植局部微环境的能力，明确GLP-1增强干细胞抗损伤的分子机制。随后选取关键旁分泌蛋白和信号通路，明确GLP-1放大ADSCs旁分泌因子表达的效能以及诱导ADSCs向心肌细胞分化的信号规律。同时利用实时活体示踪技术对细胞进行标记，阐明GLP-1干预下ADSCs在心梗部位的存活、增殖与分化规律。通过体内功能和组织学检测，最终制定并验证能够用于心肌梗死治疗的基于GLP-1的有效干细胞移植模式。

目的：心肌梗死是危害人类健康的头号杀手。常用的治疗手段如介入治疗、外科动脉搭桥术等都无法从根本上挽救已经坏死的心肌。近20年来，干细胞移植技术通过再生心肌及旁分泌功能，修复心梗后坏死损伤的组织细胞，成为改善患者心功能和临床预后的治疗方法之一。然而，人们发现限制ADMSCs在临床上进一步应用的主要难题在于心梗后移植的细胞在一周内大量死亡，从而导致了移植效率低下。其主要机制是由于移植后的细胞不耐受心梗后的氧化应激微环境，进而出现细胞凋亡坏死。因此，探讨氧化应激致细胞凋亡的关键信号通路并以此为靶点来提高细胞抗氧化应激损伤的能力是目前的研究热点。研究表明GLP-1能促进骨髓间充质干细胞的增殖、降低其调亡。因此,Exendin-4有望作为辅助干细胞治疗心肌梗死的药物。本研究的目的是研究Exendin-4辅助ADSCs移植治疗心肌梗死的效果及可行性并对其发挥作用的生物学机制以及氧化应激介导ADMSCs凋亡的关键信号通路。.结果：.1.分离培养的ADMSCs表面抗原为CD31-、CD34-、CD45-、CD29+、CD90+。2.ADMSCs能够在诱导培养基中向骨及脂肪细胞分化。3.Exendin-4能够以剂量依赖方式提高体外培养时ADMSCs的数目。4. 实验结果提示随着艾塞那肽浓度的提高，细胞迁徙能力增强。5. Exendin-4预处理后降低过氧化氢条件下凋亡细胞数目，同时上调GSH和SOD而抑制胞内ROS和MDA含量；维持线粒体正常高电位，抑制细胞色素C的渗漏；下调凋亡蛋白并提高保护性蛋白的表达。6.Exendin-4可以借由PI3K/Akt信号途径激活Sfrp2，使用LY294002阻断PI3K/Akt通路或是使用siRNA敲低Sfrp2的表达可以抑制Exendin-4对ADMSCs线粒体膜电位、凋亡相关蛋白的调控作用，逆转Exendin-4的抗凋亡作用。7.纵向在体生物发光成像显示Exendin-4促进了移植的ADSCs存活,辅助ADSCs降低了心肌的氧化应激、凋亡以及纤维化,改善了心肌活性及心功能,增强了 ADSCs向心肌细胞和血管平滑肌细胞的分化率。体外,Exendin-4降低了 ADSCs的调亡,增强了其旁分泌效应。 8.Exendin-4能降低ADSCs的ROS水平、坏死以及LDH的释放。