体内监测移植干细胞神经元分化的探索：NSE启动子引导的TYR报告基因多模态分子成像

The stem cell therapy for neural diseases, such as stroke, is the research hotspot at present. Although optical imaging, MRI imaging and molecular imaging of nuclear medicine have been widely used in tracking the transplanted stem cell, it is still beset by the difficulty of monitoring the neuronal differentiation of stem cells in vivo. Neuron-specific enolase (NSE) is an important marker of neuronal differentiation. Tyrosinase (TYR) as a new type of multimodality reporter gene, which could be applied for photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and nuclear medicine imaging respectively. Therefore, NSE promoter-driven TYR is expected to achieve monitoring the neuronal differentiation of transplanted stem cell in vivo. This study intends to construct the lentiviral vector expressing TYR reporter gene under the control of NSE promoter, and to obtain the bone marrow mesenchymal stem cells (MSCs) transfected with the vector stablely. The transfected MSCs are transplanted into the rats model of middle cerebral artery occlusion (MCAO). The method of PAI/MRI/microPET multimodality imaging is introduced to monitor the MSCs non-invasively and dynamically. Furthermore, the neural differentiation of transplanted stem cells is evaluated with the nerual specific markers, and analyzed the relationship with the result of multimodality imaging. It is expected to develop a simple, effective and non-invasive method to monitoring the neuronal differentiation of the tranplanted stem cell in the model of stroke.

干细胞治疗是脑卒中等神经系统疾病的研究热点，常用的光学成像、MRI成像和核医学分子显像多用于监测移植干细胞体内的分布、迁移和存活状态，对其神经元分化的监测尚无有效的方法。神经元烯醇化酶（NSE）是神经元分化的重要标志物，酪氨酸酶（TYR）则是一种可以同时用于光声成像（PAI）、磁共振成像（MRI）和核医学分子显像的新型多模态报告基因，以NSE特异性启动子引导的TYR有望实现对移植干细胞神经元分化的活体监测。本研究拟构建NSE特异性启动子引导的TYR基因慢病毒表达载体，稳定转染骨髓间充质干细胞，将转基因干细胞引入脑卒中模型，利用PAI/MRI/microPET多模态成像对移植干细胞进行无创、动态监测，分析多模态成像与干细胞神经元分化组织学检测结果的相关性，以期为脑卒中模型中移植干细胞神经元分化的监测寻找一种简单、有效、无创的活体监测方法。

干细胞治疗是脑卒中等神经系统疾病的研究热点，活体监测移植干细胞具有重要意义。本研究拟以酪氨酸酶基因（tyrosinase，TYR）为报告基因，转染骨髓间充质干细胞（MSCs），以18F-5-FPN为探针靶向酪氨酸酶催化产生的黑色素，通过PET/CT和PET/MR多模态显像，监测脑卒中模型中移植转TYR报告基因干细胞的分布及存活状态。首先构建TYR基因慢病毒表达载体，并转染MSCs；RT-qPCR、Western Blot、酶标仪吸光度检测法、免疫荧光法及Masson-Fontana染色法均证明，低糖培养下转基因MSCs（MSCs-V+）能稳定高表达报告基因TYR。将上述MSCs-V+通过立体定位注射法原位移植入大鼠大脑中动脉闭塞（MCAO）模型。线栓法MCAO建模的成功率为85%，立体定位原位移植法的成功率为90%，模型鼠28天存活率平均为70%。18F-FPN PET/CT及PET/MRI显示移植干细胞后第2天梗死区未见明显放射性显影，第7天后可观察到梗死区明显的放射性摄取（SUV梗死区/对侧为2.83±0.89），14天、28天时仍可见部分放射性摄取；而移植不携带TYR的MSCs组的模型鼠梗死区未见明显放射性摄取（SUV梗死区/对侧为1.42±0.29）。18F-FDG PET/CT结果显示在干细胞移植后的第7天即可观察到梗死区葡萄糖代谢的异常活跃，之后逐渐降低。研究结果表明，借助TYR/18F-5-FPN报告基因系统，通过PET/CT及PET/MRI多模态显像可以实现脑卒中模型移植干细胞的活体无创监测，联合18F-FDG PET显像还可为干细胞移植后的疗效进行动态评估。