多突变基因调控技术构建iPSCs来源的肝癌体内外模型及其机制研究

The study on the interaction mechanism of different mutation genes and their regulation methods is one of the effective ways to improve the overall survival rate of liver cancer patients, and the individualized multi-mutant gene regulation research model of liver cancer is the basis of this approach. This study is intended to demonstrate that CRISPR/Cpf1 gene editing technology can be used to individualize multiple gene mutations in patients induced pluripotent stem cell (iPSCs)-derived hepatocyte-like cells. We firstly constructed patient-derived iPSCs and put the CRISPR/Cpf1 real-time gene editing system that can regulate multi-mutation genes into it based on the specific mutation gene information obtained from functional genome sequencing, followed by their pluripotency evaluation. After induced expression of the above system, multiple gene mutations were induced in the hepatocyte-like cells of iPSCs and the hepatic organoid and humanized rat livers to form hepatoma in vitro and in vivo models, and the mutagenicity was verified. To evaluate the in vitro and in vivo models, tumor biological characteristics will be checked compared with the patient's individual liver cancer tissue consistency, and we also further clarify the molecular mechanism of action between its multiple mutations.

对不同突变基因相互作用机制及及其调控方式的研究是提高肝癌患者总体生存率的有效途径之一，而肝癌个体化的多突变基因调控研究模型是这一途径的基础。本研究拟证明：通过CRISPR/Cpf1基因编辑技术，对患者诱导多潜能干细胞（iPSCs）来源的肝细胞样细胞进行个体化的多基因突变调控，可成功构建肝癌体内外模型。本团队首先构建患者来源的iPSCs，并以功能基因组测序所获得的特定突变基因信息为依据，在iPSCs内构建可调控多突变基因的CRISPR/Cpf1实时基因编辑系统，验证其多潜能性。诱导表达上述系统后，使iPSCs来源肝细胞样细胞及其构建的肝脏类器官团培和人源化大鼠肝脏发生多基因突变，形成肝癌体内外模型，并验证致突变率。评估体外和体内模型的肿瘤生物学特性与患者个体肝癌组织的一致性，并进一步明确其多突变基因间的分子作用机制。

对不同突变基因相互作用机制及及其调控方式的研究是提高肝癌患者总体生存率的有效途径之一，而肝癌个体化的多突变基因调控研究模型是这一途径的基础。本研究拟证明:通过CR ISPR/Cpf1基因编辑技术，对患者诱导多潜能干细胞(iPSCs)来源的肝细胞样细胞进行个体化 的多基因突变调控，可成功构建肝癌体内外模型。本团队首先构建患者来源的iPSCs，并以功 能基因组测序所获得的特定突变基因信息为依据，在iPSCs内构建可调控多突变基因的CRISPR/Cpf1实时基因编辑系统，验证其多潜能性。诱导表达上述系统后，使iPSCs来源肝细胞样细胞及其构建的肝脏类器官团培和人源化大鼠肝脏发生多基因突变，形成肝癌体内外模型，并验证 致突变率。评估体外和体内模型的肿瘤生物学特性与患者个体肝癌组织的一致性，并进一步明 确其多突变基因间的分子作用机制。