BRAFV600E突变与甲状腺癌失分化及碘-131抵抗密切相关。包括我们团队在内的国内外研究发现激酶抑制剂等新型再分化策略均存在耐药问题。最新研究发现，CRISPR/Cas9技术能够实现基因组特定位点的精确和高效编辑。本课题拟利用CRISPR/Cas9技术敲除甲状腺癌细胞BRAFV600E突变基因，通过RT-PCR、Western blot、免疫荧光、流式细胞术、放射性核素摄取/流出、克隆形成等体外实验和免疫组化、小动物PET、SPECT显像、核素治疗等体内实验，在基因、蛋白和功能水平上评价敲除BRAFV600E基因后甲状腺癌细胞碘和葡萄糖代谢的变化，评价该新型基因治疗技术介导甲状腺癌精准再分化的效能并探索其机制，分析碘-131难治性分化型甲状腺癌治疗新模式（基因治疗联合碘-131治疗）的可行性，并进一步阐明BRAFV600E突变与甲状腺癌恶性表型之间的关系。

BRAFV600E gene mutation has been demonstrated to be associated with dedifferentiation and refractory to radioiodine treatment in differentiated thyroid carcinoma (DTC). Drug resistance also exists when using the current redifferentiation strategy such as kinase inhibitors to treat radioiodine-refractory DTC. The latest research found that CRISPR/Cas9 technology can achieve precise and efficient editing of the genome specific sites. The present study is designed to investigate the effect of BRAFV600E knockout using CRISPR/Cas9 on the redifferentiation and the metabolism of iodine and glucose in authenticated thyroid carinoma cell lines harboring BRAFV600E mutation in vitro and in vivo. The effect and mechanism of such novel redifferentiation strategy will be evaluated on gene, protein and functional levels using RT-PCR, Western blotting, immunofluorescence, flow cytometry, radioisotope uptake/efflux, cologenic assay, immunohistochemistry, micro-PET, micro-SPECT, and 131I therapy. Our project aims at analyzing the feasibility of the new modality (gene therapy combined with radioiodine therapy) in the treatment of radioiodine-refractory DTC, and illustrating the relationship between BRAFV600E gene mutation and malignant phenotype of DTC.