MRI分子影像报告移植干细胞的凋亡和分化研究及应用

Magnetic resonance imaging (MRI) is a widely used clinical imaging method with advantages of non invasiveness, high spatial resolution, no limit to tissue penetration depth, and variable labeling and imaging modes. MRI in vivo tracking of stem cell transplants in terms of their viability, migration and homing, and differentiation is critical for both the fundamental understanding of in vivo stem cell functions and clinical evaluations of their beneficial effects. However, the MRI methods reported so far can only provide information on cell migration and cell death. No available method can report cell differentiation. Recently, we have developed a Gd-based MRI contrast agent and a labeling strategy for long term in vivo tracking of stem cell transplants with T1/T2 dual-mode imaging effect. Pulsed electroporation is used to induce cell-assembly of Gd agents into nanoclusters for cell labeling. In vivo MRI of labeled cell transplants reveals abundant information on cell viability and physiology, in vivo migration, etc. We here propose to combine our MRI labeling strategy with specific cell physiological process to further develop a MR molecular imaging strategy for report of cell apoptosis and differentiation. Our proposal includes labeling cells with Gd agents that can be cleaved by Caspase-3 over-expressed during cell apoptosis to report cell apoptosis, and with Gd agents that can be cleaved by specific protease over-expressed in differentiated daughter cells. Pulsed electroporation induces cell-assembly of the relevant Gd agents into nanoclusters for cell labeling that results in MRI signal reduction, and cleavage of Gd agents by the relevant protease induces disassembly of the nanoclusters that induces recovery of the Gd agents into MRI signal enhancement. The switch from signal reduction to signal enhancement exhibits a significant change in MRI contrast for report of cell apoptosis or differentiation.

磁共振影像（MRI）因空间分辨率高、不受组织深度限制等优点在临床上广泛使用。MRI活体示踪干细胞移植体内后的存活、迁移及定向分化，在干细胞再生医学研究及临床疗效评价都有重要应用价值，但目前报道的技术只能提供细胞迁移及死亡的信息，不能报告细胞在体内的分化过程。前期研究中，我们利用Gd基造影剂结合电穿孔标记技术，诱导细胞自组装纳米粒子标记细胞，通过MRI实现长时间活体示踪移植干细胞，并意外获得T1/T2双模造影效果。该T1/T2双模造影模式可以提供丰富的影像学信息，揭示移植干细胞在体内的生理行为。本申请建议在前期工作基础上，结合干细胞移植到体内后的特定生理过程，研究MRI分子影像技术报告干细胞移植到体内后的凋亡、分化等过程。研究内容包括利用细胞凋亡过程过表达的Caspase-3，以及细胞分化产生的子代细胞中差异表达的剪切酶，解构形成的纳米粒子，导致MRI影像对比度变化，报告细胞的凋亡和分化。

本项目提出结合移植干细胞体内属性演变过程中呈现的特异性酶表达水平差异，实现MRI造影剂结构形态的可控变化，从而呈现T1/T2信号增强的显著差异，建立报告干细胞移植到体内后的凋亡、分化等过程的MRI分子影像技术。项目实施过程中，基于细胞凋亡过程过表达的Caspase-3及其底物多肽DEVD，设计制备了MRI探针5KDEVD-DOTA-Gd，通过电穿孔标记诱导细胞将分子探针组装为纳米探针；随后又尝试利用CBT-Cys点击化学反应自组装方法诱导细胞内自组装纳米探针；初步探索了利用纳米探针结合MRI报告移植细胞体内凋亡过程的可行性及局限性。为优化酶响应MRI纳米探针的结构以克服其局限性，进一步设计制备了分子内淬灭荧光（IQF）探针Abz-CDEVDK（Dnp），通过细胞荧光影像研究不同结构形态酶响应探针被Caspase-3酶切的效率。围绕MRI分子影像报告移植干细胞定向分化过程的课题，首先探索了神经干细胞分化为神经元和星形胶质细胞过程中，后者metrotrypsin 酶表达水平显著上调，设计制备了NapCFGKTGGd自组装纳米探针，用于报告细胞分化过程，发现细胞分化过程中细胞形态的显著变化也会导致MRI对比度的变化。在前述工作基础上，最终在细胞分化体系和酶催化体系选择上实现了突破，选择了一个分化效率高且分化速度可控的HL60细胞分化体系，随后又建立了一个基于β-环糊精接枝组装实现配位键/共价键构建纳米探针的技术路线，用于验证MRI分子影像报告细胞体内定向分化过程的可行性，目前正围绕这个细胞分化体系和技术路线开展工作。