基于CRISPR/Cas9基因组定点编辑技术的成年小鼠肝癌模型建立

Primary hepatocellular carcinoma (HCC) is one of the most common cancers in China. Animal model is an important platform which can not only help to elucidate the pathophysiology of liver cancer, but may also be employed for the development of therapeutic approaches in preclinical studies. However, substantial challenges persist in modeling liver cancer tumorigenesis, since its natural history involved different genetic alterations that ultimately lead to malignant transformation of the hepatocytes. Thus, how to take the advantage of the more efficient gene editing technology to generate more faster, simpler and more physiologic mimic liver cancer mouse models, which can model the multiple mutations simultaneously in the development of HCC, is one of the hot spots in the future research of HCC animal model. In China, more than 80% of HCC is etiologically associated with hepatitis B virus (HBV). Our previous study and other reports have showed that the high frequency of HBV intergaration in hTERT, MLL4 and CCNE1 genes, the frequent MLL4 involved chromosome translocation, and combination of multi-gene mutations are the important factors to trigger hepatocarcinogenesis in China. In this study, we will use hydrodynamic injection technique to deliver CRISPR/Cas9 genome editing tools to the liver to target and directly induce somatic mutations of multiple driver genes for HCC occurrence. The CRISPR/Cas9-mediated somatic mutations would reproduce the causative factor of HCC including HBV intergration, MLL4 chromosome translocational mutation and combined mutation of P53, Hnf-4α and Rae-1 genes. We hypothesized CRISPR/Cas9 system can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse liver cancer models, and such a system will be of practical application in the HCC biology study, clinical treatment and drug development.

肝细胞癌是我国常见的恶性肿瘤。肝癌动物模型是肝癌基础和临床研究的重要材料，但目前尚无满足理想动物模型标准的小鼠肝癌模型。如何利用更高效的基因编辑技术，模拟肝癌由多个驱动突变促发的真实情况，建立与人原发性肝癌病因相似、表型特征一致、简单易行的小鼠模型是肝癌动物模型未来研究的热点之一。我国肝癌多与乙肝病毒（HBV）感染有关。我们的前期研究和文献报道显示，HBV在hTERT、MLL4和CCNE1基因的高频整合、MLL4基因易位突变以及多基因联合突变可能是促发我国肝癌发生的重要因素。本研究拟采用CRISPR/Cas9系统和水压动力注射法，直接对成年野生型或HBV转基因小鼠肝细胞进行多基因联合编辑，模拟人肝癌发生的HBV整合、MLL4易位以及P53/Hnf-4α/Rae-1联合突变等诱因，探索在一代小鼠内建立多基因联合修饰肝癌模型的方法，为肝癌发病机制研究、临床治疗和药物研发提供理想的实验动物模型。

肝细胞癌是我国常见的恶性肿瘤。肝癌动物模型是肝癌基础和临床研究的重要材料，但由于肝癌的病因复杂，目前尚无满足理想动物模型标准的小鼠肝癌模型。如何利用更高效的基因编辑技术，模拟肝癌由多个驱动突变促发的真实情况，建立与原发性肝癌病因相似、表型特征一致、简单易行的小鼠模型是肝癌动物模型未来研究的热点之一。本课题旨在探索一种基于尾静脉水压动力注射和CRISPR/Cas9系统的快速简便的小鼠肝癌模型建立方法。我国肝癌多与乙肝病毒（HBV）感染有关，P53基因突变和PTEN基因失活是乙肝相关肝癌中常见的两种遗传事件。本课题通过水压动力注射技术将靶向小鼠肝细胞的p53与Pten基因的CRISPR/Cas9系统递送至HBV转基因小鼠的肝细胞，并观察小鼠肝癌的发生情况。结果表明，HBV转基因小鼠在sg-p53/Pten双表达质粒尾静脉水压动力注射4个月后即可形成明显肝肿瘤，而野生型小鼠则在10个月时才出现较小可见肿瘤。进一步分析发现，sg-p53/Pten质粒注射6个月和8个月的HBV转基因小鼠的肝重体重比明显高于对照组。免疫组化检测结果显示此方法诱导的肿瘤组织中存在明显的谷氨酰胺合成酶异常表达以及p53和Pten表达降低，且肝细胞中脂肪堆积明显。PCR联合测序实验证实在所检测的肿瘤结节中均有p53和Pten基因突变，提示肿瘤的发生是由于p53和Pten基因突变引起的。进一步对肿瘤组织进行RNA-Seq分析结果显示，我们构建的小鼠肝癌模型与人肝细胞癌的转录谱非常相似，尤其是与PTEN失活的人肝癌组织转录谱具有高度相似性。此外，我们还探讨了Ubxn8与Pten、Cxcl2与p53 / Pten等多基因的联合敲除，以及HBV整合小鼠肝癌模型的建立。总之，本研究建立的小鼠肝癌模型与我国肝癌发生病因相似，简单经济，且在一代小鼠内即可完成，将为我国肝癌的发病机制研究、临床治疗和药物研发提供理想的实验动物模型。