Tet-on调控报告基因酪氨酸酶用于光声、PET及MRI多模态成像的探索研究

Multimodality molecular imaging is a new approach using various combinations of different imaging modalities to provide rich information of biological process. However, for both direct and indirect imaging strategy, most of the multimodality imaging agents reported so far is complicated constructed and needed to add different functional groups. It is very important to obtain a simple molecular probe with easily construction. In this study, Tyrosinase (TYR) will be used as a multifunctional reporter gene. TYR is a key enzyme in melanin synthesis. After transduction of tyrosinase gene into cells and encoding an active tyrosinase, melanin could synthesis. Based on photoacoustic effect, photoacoustic imaging could be achieved for melanin could absorb light energy. PET imaging could be obtained because melanin could bind with 18F-labeled benzamide specifically. MRI imaging could be obtained based on the characteristic that melanin could combine with iron due to a large number of anion on its surface. Moreover, in order to avoid the unnecessary physiological role after the reporter gene expresses, Tet-On will be used to control the expression and quantity of TYR precisely. The aim of this study is to establish a new reporter gene system tyrosinase under the control of Tet-On gene. The establishment of this system not only combines photoacoustic imaging, a new imaging modality, with other molecular imaging modalities, which could solve the problem of spatial resolution and sensitivity, but also provides high spatial resolution and velocity resolution imaging of blood flow in vivo by Doppler photoacoustic imaging. This system will have much impact on the multi-modality molecular imaging based on indirect strategy, and have important implications.

在多模态分子影像中，基于直接及间接显像策略的分子探针构建较为困难，构建简单易得分子探针十分重要。 本研究应用酪氨酸酶作为多功能报告基因用于多模态显像。酪氨酸酶是黑色素生成的关键限速酶，将其导入细胞后可生成黑色素。黑色素能吸收光能并通过光声效应，实现光声成像；黑色素可与18F-标记苯酰胺类似物特异性结合，可实现PET显像；黑色素带有大量阴离子，可结合铁并实现MRI显像；研究还应用Tet-On基因精确控制和调节酪氨酸酶的表达及产量，以避免应用报告基因可能出现的不必要的生理作用。 本研究目的构建Tet-On调控的酪氨酸酶作为报告基因，在精确调控其表达及产量的基础上，应用单一报告基因达到同时进行光声、PET和 MRI多模态分子显像的目的。这一系统的建立不仅将光声成像这一新型显像模态与其他分子影像融合起来，而且将对基于间接策略的多模态分子探针的构建产生巨大的影响，这一创新性研究具有良好转化应用前景。

本研究应用酪氨酸酶作为多功能报告基因用于多模态显像。酪氨酸酶是黑色素生成的关键限速酶，将其导入细胞后可生成黑色素。黑色素能吸收光能并通过光声效应，实现光声成像；黑色素可与18F-标记苯酰胺类似物特异性结合，可实现PET显像；黑色素带有大量阴离子，可结合铁并实现MRI显像；研究还应用Tet-On基因精确控制和调节酪氨酸酶的表达及产量，以避免应用报告基因可能出现的不必要的生理作用。.本研究结果显示黑色素特异性探针18F-5-FPN标记率为5%～8%，其放化纯大于95%，比活度为100～120GBq/μmol，具有与黑色素细胞B16F10特异性结合的能力，18F-5-FPN探针PET显像对应肿瘤/背景比值明显高于18F-FDG探针10倍左右，能及时探测到B16F10肺转移模型内微小转移病灶。TYR报告基因通过转染，可在非黑色素 MDA -MB -231细胞中诱导黑色素的产生。Tet-on系统可以精确调控报告基因表达及产量。细胞体外研究证实了单一报告基因TYR进行MRI、PET 和PAI 多模态显像的可行性。用慢病毒包装TYR转染至大鼠骨髓间充质干细胞（MSCs），构建稳定表达酪氨酸酶的TYR-MSCs细胞系，具有特异性摄取18F-5-FPN的能力，并具有细胞水平MRI和PAI成像能力，建立大鼠心梗模型并行TYR-MSCs移植后，可利用PET、MRI、PAI进行活体三模态示踪移植干细胞。