建立基于人胚胎干细胞的新型胰腺癌模型来研究癌变机制

Pancreatic adenocarcinoma (PDAC) is one of the most lethal malignances due to its limited response to current antitumor treatments. Novel therapies are urgently needed. The key to solving this problem is to establish humanized disease model closely related to human PDAC physiology and pathology. Previously, our group have generated TP53 conditional knockout human embryonic stem cell line, denoted TP53CKO hESCs. On this basis, we will generate TP53CKO:KRAS(G12D)CKI hESCs by knockin conditional expressing KRAS(G12D), which is a constitutively active form of KRAS, to the endogenous KRAS locus using CRISPR/Cas9-based gene targeting. Once TP53CKO:KRAS(G12D)CKI hESCs are differentiated to pancreatic progenitor cells, TP53 will be knockout and KRAS(G12D) will be expressed by Cre/LoxP system, and the cells will be transplanted into NOD-SCID mice to measure the capacity for spontaneous PDAC tumorigenesis. Based on our novel humanized PDAC model, we will further identify the genomic DNA mutations as well as gene expression changes during PDAC progression, exploring the downstream cancer-related pathways and their regulation networks, and finally demonstrate the effects of the corresponding small molecular drugs on the treatment of PDAC. By closely combining regenerative medicine and tumor targeted therapy, these findings will be instrumental for demonstrating the molecular mechanism of PDAC tumorigenesis, and will provide novel pre-clinical humanized model for PDAC drug discovery.

胰腺癌是恶性程度极高、预后极差的肿瘤，针对胰腺癌的新药研发一直以来都是困扰医学界的重大问题。解决这一问题的关键是开发新的与人生理密切相关的胰腺癌研究模型。本课题组前期已建立TP53条件性敲除的人胚胎干细胞TP53CKO hESCs，在本项目中，我们将采用CRISPR/Cas9技术在该细胞中敲入条件性表达的激活型KRAS(G12D)，通过Cre/LoxP技术在TP53CKO:KRAS(G12D)CKI hESCs分化来源的胰腺前体细胞中敲除TP53及表达KRAS(G12D)，原位移植至免疫缺陷小鼠建立新型胰腺癌模型；并以此为基础通过高通量测序研究在胰腺癌发生发展过程中的基因组DNA突变及基因表达改变，探索其调控网络及作用机制，研究相应靶向药物的疗效。本研究将通过紧密结合再生医学及肿瘤精准医学为胰腺癌研究提供全新的人源化模型，并为阐明胰腺癌的发生发展机制及研发新型有效的胰腺癌治疗策略奠定基础。

代谢通路广泛参与了人胚胎干细胞自我更新及分化的命运调控过程。我们的研究发现，p53敲除能显著抑制胚胎干细胞向中内胚层的分化，并促进人胚胎干细胞的有氧糖酵解且抑制其氧化磷酸化。通过代谢组学分析，我们进一步发现，在胚胎干细胞自我更新及中内胚层分化过程中，p53敲除均能显著下调总胆碱类代谢产物，同时上调磷脂酰胆碱类代谢产物，表明p53参与调控了人胚胎干细胞中的胆碱代谢通路。综上，本研究表明p53在调控三大代谢通路中糖代谢、脂代谢以及人胚胎干细胞命运决定过程中发挥了至关重要的作用。