靶向多肽T1型造影剂用于磁共振影像活体示踪间充质干细胞研究

Application of stem cell transplantation in tissue regeneration is an important research field of regenerate medicine.In vivo magnetic resonance imaging (MRI) of transplanted stem cells in terms of their viability, migration and homing, differentiation, response to various endogenous stimuli is critical for both fundamental understanding of stem cell functions and clinical evaluation of their beneficial effects. MRI tracking of stem cells requires labeling of the cells with contrast agent which allows them to be distinguished from the in vivo background. Superparamagnetic iron oxide nanoparticles have been used to label various stem cells, which can track cell migration with in vitro sensitivity of single cell and in vivo sensitivity of hundred(s) of cells, but yields no information on cell viability. We propose in the current application to develop a novel type of T1 contrast agent for labeling of mesenchymal stem cells (MSCs) and in vivo MRI tracking of MSCs in terms of their viability. The contrast agent is a coupling complex of Gd-DOTA, MSC-specific peptide, and dendrimers which combines several strategies to enhance its T1 contrast effect. We aim to establish a strategy to reveal the viability of transplanted MSCs by labeling MSCs with this novel contrast agent and by tracking them under high-field and high-gradient MRI in vivo.

基于干细胞移植的组织再生工程是再生医学的重要研究领域。利用磁共振影像技术研究移植干细胞在体内的维持与分化过程，对人们认识干细胞在体内的迁移、繁殖、分裂与分化等过程具有不可替代的作用。目前利用各种商业或自制的基于超顺磁氧化铁纳米粒子的T2型造影剂标记干细胞，在高场条件下已经可以实现体外单细胞、体内百个细胞的影像灵敏度，但这种标记只能示踪细胞在体内的迁移，无法区分细胞在体内的死活状态。本项目拟结合高场（高灵敏度）、高梯度（高空间分辨率）磁共振影像技术、多肽靶向结合技术、以及各种增强钆络合物造影效果（对比度）的技术，探索研究一种基于Gd-DOTA和靶向多肽分子的T1型磁共振造影剂，用于间充质干细胞磁标记，再利用高场（11.7T）影像技术提供的高灵敏度和高梯度带来的高空间分辨率，建立能够区分间充质干细胞在活体内的死活状态的影像方法。

本课题实施过程中，系统研究了多肽Gd基MRI造影剂标记间充质干细胞的效率及其造影效果。利用孵育和电穿孔两种方法都实现了标记效率达到1010 Gd/细胞量级，通过TEM观察造影剂与细胞界面结合状态，揭示了游离态、细胞膜结合及纳米簇等三种不同结合状态；建立磁共振造影剂体外定量评估的标准实验步骤，研究MRI造影剂与细胞界面作用机制，揭示了三种不同界面结合状态的造影剂对细胞水质子的T1和T2弛豫速率的影响机制及MRI造影效果存在显著不同。研究发现利用多肽Gd基造影剂和电穿孔技术标记细胞时，会诱导细胞将造影剂组装成纳米簇进入细胞质中，显著延长造影剂与细胞结合时间，并使细胞呈现显著的MRI信号减弱效应。从这个发现出发，开发了一种多肽Gd基T1/T2双模造影剂，建立了以裸鼠为动物模型，利用MRI示踪多肽Gd基造影剂标记的间充质干细胞移植体内后命运（细胞迁移、死活状态）的实验方法及影像分析方法。