运用基因打靶和CRISPR/Cas9技术构建原发性胆囊癌小鼠模型

Primary gallbladder carcinoma is the most common malignancy of the biliary tract and the prognosis is very poor. Whole-exome sequencing of gallbladder carcinoma identifies recurrent mutations in TRP53, KRAS and ERBB3. Establishment of primary gallbladder carcinoma mouse models through gene targeting technique is vital for the investigation of tumor pathogenesis. However, up to now, the established mouse models of gallbladder carcinoma only include transplanted tumor model and Erbb2 transgenic mouse model. Those are not enough for the gallbladder carcinoma pathogenesis research. This project aims to make gallbladder epithelial cell-specific Cre recombinase-expressing mice, and to establish a mouse model of gallbladder carcinoma through inactivation of Trp53 and activation of Erbb3 in gallbladder epithelial cells. Then, we will further study the molecular mechanism of Trp53 and Erbb3 in gallbladder carcinoma pathogenesis. Moreover, we will apply CRISPR/Cas9 technique to establish a mouse model by inactivation of Trp53 and activation of Kras in gallbladder.

原发性胆囊癌是胆道系统最常见的恶性肿瘤，预后极差。胆囊癌全外显子组测序发现TRP53,KRAS和ERBB3是反复出现突变的基因。运用基因打靶技术建立原发性胆囊癌小鼠模型，对于研究肿瘤的发生机理至关重要。然而，到目前为止，胆囊癌的小鼠模型仅有移植瘤模型和Erbb2转基因小鼠模型，不能满足胆囊癌发生机理研究的需求。本研究拟制作胆囊上皮细胞特异表达Cre重组酶的小鼠，然后运用该小鼠在胆囊上皮细胞中特异敲除Trp53基因和特异活化Erbb3基因，制作小鼠原发性胆囊癌模型，并进一步研究Trp53和Erbb3在胆囊癌发生过程中的分子机理。此外，我们还将运用CRISPR/Cas9技术在小鼠胆囊上皮细胞中敲除Trp53基因并活化Kras基因，制作胆囊癌小鼠模型。

原发性肿瘤小鼠模型的建立对于肿瘤发生机理的研究至关重要，本项目旨在建立原发性胆囊癌小鼠模型。我们构建了Tm4sf4-CreERT2基因敲入小鼠，在该小鼠胆囊上皮细胞中表达CreERT2，经他莫昔芬诱导，可重组loxp位点，实现组织特异性基因修饰。在小鼠（Tm4sf4-CreERT2;KrasG12D/+;Trp53R172H/+）胆囊上皮中表达Trp53和Kras基因的突变体后，胆囊壁增厚，但没有出现胆囊癌。在小鼠胆囊中用腺相关病毒AAV9介导的Trp53、Kras和Pten基因编辑导致胆囊肉瘤快速生长，未发现胆囊癌。此外，我们发现桦木酸通过抑制SCD1的表达抑制胆囊癌细胞的增殖并促进细胞凋亡。