Development of humanized NOG mice reconstituted with human hepatocytes and their applications in drug discovery and infectious disease research.

本研究旨在建立药物评价模型和感染性疾病模型，目的是研制一种具有人肝细胞的重组小鼠。本研究分为以下三个步骤，每一步的研究结果如下：第一步：建立持续性肝损伤转基因小鼠，建立携带单纯疱疹病毒胸苷激酶基因的转基因小鼠(Alb-HSV-TK)，通过白蛋白增强子和启动子诱导更昔洛韦(GCV)诱导肝细胞特异性损伤。通过基因显微注射实验获得208只仔鼠，经聚合酶链式反应基因分型试验获得9只初筛呈阳性反应的方正小鼠。第二步：用GCV诱导肝细胞特异性损伤。根据结果，只有TG-Alb-HSV-TK#7-2株(TG-TK#7-2)的AST和ALT水平升高。此外，TG-TK#7-2显示耳朵有明显的黄疸。对小鼠肝脏的组织学检查发现了一些异常，包括肝细胞游动和肝细胞巨噬细胞增多。第三步：应用于人肝细胞重组小鼠的药物评价研究。不幸的是，第三步无法在计划的时间内完成。主要原因是TG-TK#7-2系的不育性，使得使用体外受精和胚胎移植技术进行大规模生产变得不可能，并极大地推迟了我们的生产计划。然而，我们获得了一些TG-TK#7-2 NOG小鼠，并已经开始了一项人类肝细胞移植实验，每周评估一次人源化迹象(人肝NOG)。人肝NOG小鼠将成为评价药物疗效和药代动力学的有用模型。

The aim of this study, which was designed to establish a drug evaluation model and infectious disease model, was "to develop a reconstituted mouse with human hepatocytes". The research plan consisted of the following three steps, and the results at each step are described.First step : Development of genetically modified mice with sustained hepatic injury.We established a transgenic mouse (Alb-HSV-Tk), which carries the herpes simplex virus thymidine kinase gene with an albumin enhancer and promoter, to induce hepatocyte specific cell injury by ganciclovir (GCV). Two-hundred and eight pups were obtained by DNA microinjection experiments and we succeeded in obtaining 9 founder mice that showed positive reactions in the first screening by the PCR genotyping test.Second step : Induction of hepatocyte specific cell injury by GCV administration.Transgenic pups were obtained from nine Alb-HSV-Tk founders, and the ability to induce hepatic injury by GCV injection was examined. According to the results, elevated levels of AST and ALT were observed only in the Tg-Alb-HSV-Tk #7-2 line (Tg-Tk#7-2). Furthermore, Tg-Tk#7-2 showed apparent jaundice in the ears. Histological investigation of the mouse liver revealed a number of abnormalities, including hepatocyte vaculation and hepatocyte megalocytosis.Third step : Application in a drug evaluation study with reconstituted mice with human hepatocytes.Unfortunately, the third step could not be completed within the planned period. The main reason was the sterility in the Tg-Tk#7-2 line, which made large-scale production using in vitro fertilization and embryo transplantation techniques impossible, and drastically delayed our production plan. However, we obtained a few Tg-Tk#7-2 NOG mice, and have already started a human hepatocyte transplantation experiment with weekly evaluations for signs of humanization (Hu-Liver NOG). The Hu-Liver NOG mouse will be a useful model to facilitate the evaluation of drug efficacy and pharmacokinetics.