超声微泡靶向破坏促CCN1基因修饰的MSC归巢治疗急性心肌梗死的研究

Successful stem cell therapy after acute myocardial infarction is hindered by lack of engraftment of sufficient mesenchymal stem cells at the site of injury. Mesenchymal stem cell homing includes two-step progress: migration and colonization. Many Studies have demonstrated that ultrasound-targeted microbubble destruction could improve the colonization of mesenchymal stem cells from ischemic myocardial capillaries to infarcted myocardium, but not solved the migration from peripheral vessels to ischemic myocardial capillaries. Our new research finds that up-regulated expression of CCN1 can improve directional migration of mesenchymal stem cells from peripheral vessels to ischemic myocardial capillaries. It reveals that CCN1 will take as a target for regulation of MSC migration. Thus, we hypothesize that ultrasound-targeted microbubble destruction improves migration and homing of mesenchymal stem cells and cardiac function in myocardial infarction of rats. In this study, we seek to test the hypothesis on the basis of microbubble preparation, as follows. Studying CCN1 modified mesenchymal stem cell’influence on ventricular remodeling and cardiac function after its homing at infarcted myocardium mediated by ultrasound-mediated microbubble destruction in rat Models of acute myocardial infarction. And illuminate the molecular mechanism of MSC migration regulated by CCN1. The research will provide a new target for the directional migration of mesenchymal stem cell as well as a new idea for improving the shortcoming of mesenchymal stem cell homing through ultrasound-targeted microbubble destruction.

骨髓间充质干细胞（MSC）归巢率低是其治疗急性心肌梗死的主要障碍。静脉输注的MSC归巢包括迁移和定植两个步骤。研究证明，超声微泡靶向破坏可促进MSC从缺血区毛细血管到心肌梗死局部的定植。但这种细胞递送方式无法解决MSC从外周血管到缺血区毛细血管过程中的迁移不足，MSC归巢仍受到限制。我们最新发现，通过上调CCN1基因表达能促进MSC迁移，提示CCN1可作为调控MSC迁移的靶点。因此，我们提出假设：超声微泡靶向破坏联合CCN1基因修饰可显著增加MSC归巢，提高急性心肌梗死疗效。为此，本项目在已有微泡研究基础上，拟建立大鼠急性心肌梗死模型，应用超声微泡靶向破坏技术将CCN1基因修饰的MSC归巢大鼠缺血心肌局部，评价其归巢率及对心室重塑和心功能的影响，并阐明CCN1调控MSC迁移的分子机制。通过本课题的研究，有望为改善超声微泡靶向破坏介导MSC归巢的不足提供新思路，也为MSC定向迁移提供新靶点。

骨髓间充质干细胞归巢率低是其治疗急性心肌梗死的主要障碍。为此，探索能减少干细胞在外周损耗，有效增加其归巢率是当前干细胞移植治疗急性心肌梗死亟待解决的问题。超声微泡靶向破坏可促进MSC从缺血区毛细血管到心肌梗死局部的定植。但这种细胞递送方式无法解决MSC从外周血管到缺血区毛细血管过程中的迁移不足骨髓间充质干细胞归巢仍受到限制。我们假设，超声微泡靶向破坏联合CCN1基因修饰可显著增加骨髓间充质干细胞归巢，提高急性心肌梗死疗效。为验证假说，体外采用薄膜水化和机械振动法制备超声微泡，构建CCN1质粒，应用超声微泡靶向破坏技术介导CCN1基因转染骨髓间充质干细胞，利用细胞划痕实验和Transwell 细胞迁移实验检测CCN1 调控骨髓间充质干细胞迁移能力，应用RT-PCT、Werstern-blot研究CCN1 调控骨髓间充质干细胞迁移的分子机制；体内构建心肌梗死模型，利用超声微泡靶向破坏技术诱导CCN1 修饰的骨髓间充质干细胞进入大鼠缺血心肌局部，利用超声心动图、病理染色、免疫组化及小动物生物发光成像等技术研究其对心室重构及心功能影响。结果显示：制备微泡粒径均一、性质稳定，具有生物安全性和良好的显影效果；CCN1转染骨髓间充质干细胞后，可增加其迁移能力，这与下游基因CXCR4，PI3K和Akt过表达密切相关；超声微泡靶向破坏联合CCN1修饰可明显增加骨髓间充质干细胞归巢率，抑制心肌凋亡，减少梗死面积，促进血管新生，改善心功能，这得益于细胞因子VCAM-1，VEGF和SDF-1表达增加。CCN1修饰可显著增加骨髓间充质干细胞迁移能力，联合超声微泡靶向破坏技术可显著增减骨髓间充质干细胞归巢率，促进组织修复，改善心功能。本研究为超声介导微泡破坏技术促骨髓间充质干细胞归巢不足提供了新的思路，也为骨髓间充质干细胞定向迁移提供了新的靶点。