报告基因标记的miRNA-122肝脏表达小鼠模型的建立及其体内成像

The research process of transcriptional regulation and target validation of miRNA-122in vivo as well as the design of small molecule drugs targeting of it was hindered by the lack of convenient tools. That is a proper animal model to evaluate the regulation of the miRNA-122expression and the miRNA-122 targeting drugs. In this study, we will construct the "miRNA-122-Gluc" and the "Fluc-miRNA-122 sensor" expression vector. And then combined with the hydrodynamic-based tail-vein transfection and the phage ?C31integrase system, thus, by monitoring the luciferase reporter gene expression we can know themiRNA-122expressionlevelin the mouse model. And then, by means of the in vivo imaging we can monitor the real-time dynamic expression levels oflivermiRNA-122, then, we can study the miRNA-122expression regulation, miRNA-122targetvalidation and the evaluation of the efficiency of drugs targetingmiRNA-122. All of these will promote the research on the drug development of miRNA-122 and its function on the whole animal levels. Up to now there are no domestic or abroad reports about this model, which is the innovation of this idea and methodology.

缺乏方便的动物体内肝脏miRNA-122表达调控、以及靶向miRNA-122药物评价手段，阻碍了miRNA-122体内转录调控、靶标验证，以及以miRNA-122为靶点设计小分子药物的研究进程。对此本研究拟构建"miRNA-122-Gluc"和"Fluc-miRNA-122 sensor"表达载体，组合高压尾静脉转染技术和噬菌体整合酶系统，建立以荧光素酶报告基因来反映miRNA-122表达水平的小鼠模型，进而通过活体荧光成像技术实时动态监测肝脏miRNA-122 表达水平，用以体内miRNA-122表达调控的研究、miRNA-122靶标验证以及靶向miRNA-122药物体内评价，可从整体动物水平上促进对肝脏miRNA-122功能的研究和药物的研发。目前在国内外还未见文献报道，此点属研究思路和方法学上的创新。

由于缺乏方便的动物体内肝脏miRNA-122 表达调控、以及靶向miRNA-122 药物评价手段，从而阻碍了miRNA-122 体内转录调控、靶标验证，以及以miRNA-122 为靶点设计小分子药物的研究进程。对此本研究构建了"miRNA-122-Gluc"和"Fluc-miRNA-122 sensor"表达载体；建立了眼底静脉丛水动力转染技术，将大量含有目的基因表达质粒的盐水溶液从眼底静脉快速注入实验动物体内，实现外源基因在肝脏特异、高效表达，通过条件摸索，首先确立了应用该技术的最适转染条件，并分析了其对实验动物器官机能的影响，在转染效率方面，眼底静脉丛水动力转染技术与经典的尾静脉水动力转染技术相当；进一步，组合高压眼底静脉丛转染技术和噬菌体整合酶系统，建立以荧光素酶报告基因来反映miRNA-122 表达水平的小鼠模型，进而通过活体荧光成像技术实时动态监测肝脏miRNA-122 表达水平，用以体内miRNA-122 表达调控的研究、miRNA-122 靶标验证以及靶向miRNA-122 药物体内评价，可从整体动物水平上促进对肝脏miRNA-122 功能的研究和药物的研发。