利用CRISPR/Cas9突变HBx小鼠肝脏p53和Kras基因建立肝细胞癌模型及多基因协同作用研究

Hepatocellular carcinoma (HCC) is one of a disease which cause serious hazard to human health. The process and development of HCC are the result of the interation of multi-factors. HBx is considered to be closely related to Hepatitis B Virus (HBV) infection-induced HCC. The mutations of tumor suppressor genes and proto-oncogenes (e.g. the mutations of p53 and Kras) are important factors for the development of HCC. Currently, the synergistic effect and molecular mechanisms of the p53 and Kras mutations together with HBx in the process of HCC are still not fully understood. The lack of appropriate HCC animal models is the major bottleneck toward this issue. This project precisely mutate p53 and Kras genes through delivering CRISPR/Cas9 into the liver of HBx transgenic mice by using hydrodynamic injection in order to establish somatic mouse cancer models mimicking the somatic nature of HCC progression. The speed and degree of HCC among the HBx transgenic mice, HBx transgenic mice with p53 mutation and HBx transgenic mice with p53 and Kras double mutation will be compared by using the methods of microCT, pathology, immunohistochemistry. This may reveal the synergistic effect of p53 mutation or p53 and Kras double mutations together with HBx during the process of HCC. This study not only helps clarify the molecular mechanisms and signaling pathways in the development of HCC induced by the synergistic effect of multigene, but also provides new clues for clinical therapeutic targets. These HCC mouse models have well application prospect in the development of anti-cancer technologies and drugs as well.

肝细胞癌是严重危害人类健康的疾病之一，其发生发展是多种因素相互作用的结果，包括p53、Kras突变和乙肝病毒HBx都与肝细胞癌发生相关。目前HBx与该两基因突变在肝细胞癌发生过程中的协同作用和分子机制尚不确定，其瓶颈是缺乏合适的动物模型。本项目拟用高压水动力法运送CRISPR/Cas9到HBx转基因小鼠肝脏中，精确突变p53和Kras基因（p53R246S和KrasG12D），实现基因编辑体细胞构建肝细胞癌小鼠模型。通过影像学、病理学、免疫组化等方法比较HBx小鼠、p53突变的HBx小鼠和p53/Kras双突变的HBx小鼠诱发肝细胞癌的速度与程度，从而揭示此过程中p53突变或者p53/Kras双突变协同HBx的作用方式。研究有助于阐明多基因协同诱导肝细胞癌发生发展的分子机制和信号通路，为发现临床治疗靶标提供新的理论线索,构建的肝细胞癌小鼠模型在抗癌技术与药物研发等方面亦具有良好的应用前景。

肝细胞癌是严重危害人类健康的疾病之一，其发生发展是多种因素相互作用的结果。构建合适的肝细胞癌动物模型，将有助于深入探究肝细胞癌发生发展的分子机制。利用高压水动力法运送CRISPR/Cas9到活体小鼠肝脏可实现对小鼠肝脏基因精确编辑；相比基于生殖细胞系的基因编辑小鼠模型，该方法可在子一代中实现多基因修饰的模型构建，可加快造模时间并提高造模效率。本研究先设计了用于敲除小鼠p53和MAD2L1基因的靶点，并构建了定点敲除小鼠p53和MAD2L1基因的质粒。利用该质粒系统先在小鼠NIH/3T3中对p53和MAD2L1基因进行精确敲除，建立了阳性筛选基因敲除细胞和敲除p53和MAD2L1基因的体系。进而通过高压水动力法运送敲除MAD2L1基因和敲除p53/MAD2L1基因的质粒到C3H/HeN小鼠肝脏中，10-12个月后观察到实验组小鼠肝脏有肿瘤病变的发生。HE染色结果提示MAD2L1基因敲除组小鼠肝癌细胞核大、深染呈团片及梁状排列，癌细胞浆内可见水样变性，未见明显核分裂现象。p53和MAD2L1基因双敲除组小鼠肝癌细胞核大、深染呈团片排列，癌细胞呈多形性，可见核分裂现象。免疫组化结果显示，MAD2L1基因敲除、p53和MAD2L1基因双敲除组Ki67和CD34均呈阳性，但p53和MAD2L1基因双敲除组阳性率高于MAD2L1基因敲除组。p53和MAD2L1抗体免疫组化结果提示，MAD2L1基因敲除、p53和MAD2L1基因双敲除组分别观察到有细胞缺失表达MAD2L1、p53和MAD2L1基因。基因修饰分析结果进一步证实了p53和MAD2L1基因发生敲除。总之，本研究建立了肝细胞癌小鼠模型，证实p53和MAD2L1基因对肝细胞癌的发生和发展具有协同作用，同时敲除p53和MAD2L1基因可加速肝细胞癌的发生和发展。项目发表了论文3篇，其中SCI论文1篇，核心期刊论文2篇，申请专利1项。相关研究结果获得了2018年第三十届上海市优秀发明选拔赛银奖。后续研究仍在继续中，将进一步深入精细化分析所构建的小鼠肝细胞癌模型，以及探究其它因子与肝细胞癌发生发展的关系，相关数据正在整理中。以上研究有助于阐明多基因协同诱导肝细胞癌发生发展的分子机制和信号通路，构建的肝细胞癌小鼠模型可能在抗癌技术与药物研发等方面具有应用前景。