UTMD促MSCs移植靶向归巢与定向分化治疗心肌梗死的实验研究

Bone marrow-derived mesenchymal stem cells (MSCs) are ideal seed cells for the cell transplantation therapy of heart failure after acute myocardial infarction (AMI). The bottleneck problem of this research currently includes: the undefined proliferation of MSCs in vivo and the difficulty of homing into the injured myocardium after AMI. This project includes the combination of molecular ultrasound imaging and contrast technique could provide the possibility to early diagnose and intervene AMI. Based on our previous original results, we will carry on further study to solve above mentioned problems. This study aims to explore the potential use of ultrasound targeted microbubble destruction (UTMD) technique to achieve the following two goals: ①establishment of the nonlinear mathematical model of acoustic scattering for nanometer-microbubbles (NB) to achieve NB’s sensitive imaging in tissue or vascular structure; and use UTMD to mediate non-viral transfection of MSCs with prolyl hydroxylase domain(PHD2) shRNA plasmids, so that transfected MSCs can survival better in the hypoxic environment to exert their functions; ②use UTMD to increase the permeability of capillaries, so that the homing of MSCs injected through the tail vein to the ischemic cardiac region can be enhanced. Eventually, these manipulations should decrease myocyte death, increase angiongenesis and new myocardium formation and hence improve cardiac function. This study will shed lights on effective and noninvasive stem cell transplantation methods into infracted hearts and provide the experimental basis for such treatment.

骨髓间充质干细胞(MSCs)移植是急性心肌梗死(AMI)后慢性心衰一种具有重要潜力治疗方法。目前存在主要问题是，在持续心肌缺血状态下MSCs靶向归巢困难和分化方向不确定。课题组前期实验中，利用超声靶向微泡破裂(UTMD)介导shPHD2转染至MSCs后，可明显增强其在缺氧环境下的存活能力，并首次揭示其对MSCs向心肌分化具有调控作用。据此, 我们设想UTMD介导基因转染可提高MSCs周围定向分化因子的稳定浓度，协同增加心肌归巢能力。本项目拟: ①研究纳米微泡在组织结构中的非线性声学散射模型，优化其标记MSCs条件，对移植后的干细胞在体示踪的同时，能显著增强MSCs在缺氧环境下的存活及向心肌定向化能力； ② 建立小鼠AMI模型，通过UTMD技术实现MSCs向缺血缺氧心肌靶向归巢，最终达到减少心肌死亡，促进新血管和新心肌的形成，为临床MSCs心脏移植提供新思路和优化策略。

骨髓间充质干细胞(BMSCs)移植是急性心肌梗死(AMI)后慢性心衰的重要治疗方法，在持续心肌缺血状态下BMSCs靶向归巢困难，超声靶向微泡破坏技术(UTMD)则可以促进BMSCs精确输送到缺血心肌部位。然而，移植干细胞缺血条件下存活率低是制约干细胞治疗缺血性心肌病的重要因素。我们假设，PHD2-shRNA修饰可显著增加BMSCs活性，联合UTMD可显著提高急性心肌梗死疗效。本项目采用薄膜水合法制备新型微泡，构建PHD2-shRNA质粒，运用UTMD技术介导PHD2-shRNA体外转染BMSC，应用RT-PCR、Werstern-blot等研究PHD2-shRNA调控BMSC的耐受缺氧分子机制；建立AMI模型，并采用UTMD 技术诱导PHD2-shRNA饰的BMSC移植治疗AMI，检测PHD2 shRNA对BMSCs的存活率、心肌凋亡、血管新生以及心功能等。结果显示（1）成功制备了阳离子微泡，其具有良好的超声成像能力；（2）成功构建PHD2-shRNA质粒，UTMD介导shPHD2体外转染BMSCsPHD2 shRNA转染BMSC可通过旁分泌机制抑制心肌凋亡；（3）构建大鼠AMI模型， UTMD介导shRNA修饰BMSC移植后，可提高BMSC活性，减少心肌凋亡，缩小梗死面积，增加梗死区血管密度，改善心功能。综上，UTMD技术递送PHD2-shRNA修饰的BMSCs可促进移植细胞归巢和活性，增加缺血心肌血管新生，改善心功能。本项目研究提高我们对UTMD技术在BMSC移植中作用的认识，也为提升急性心肌梗死后干细胞治疗的有效性提供了有前景的策略。