细胞毒性T淋巴细胞相关抗原4(CTLA4)敲除和人源化小鼠模型的建立

Establishment of CTLA4-knockout mice and CTLA4-humanized mice

目的建立细胞毒性T淋巴细胞相关抗原4(CTLA4)基因人源化小鼠模型,为靶向CTLA4的肿瘤治疗性抗体研发和筛选提供有效的小鼠模型。方法利用CRISPR/Cas9技术,建立CTLA4基因人源化和敲除小鼠模型,利用PCR、RT-PCR、Western Blot、免疫组织化学和流式细胞术的方法对其进行鉴定及分析。将小鼠黑色素瘤细胞株(B16)注射到CTLA4人源化小鼠皮下,观察腹腔注射CTLA4单克隆抗体伊匹木(ipilimumab)的抗肿瘤效果。结果CTLA4人源化小鼠可以稳定表达人源CTLA4,而不表达鼠源CTLA4。CTLA4人源化小鼠出生后6个月内正常存活,组织病理学及免疫系统未见明显异常。同时在CTLA4人源化小鼠中,伊匹木减缓肿瘤生长速度。CTLA4基因敲除小鼠不表达CTLA4基因,在出生后3~5周内由于自身免疫疾病死亡。结论同时建立了CTLA4人源化和敲除小鼠。CTLA4人源化小鼠可用于筛选与CTLA4基因相关的治疗性抗体或药物。

Objective To establish a cytotoxic T-lymphocyte-associated protein 4(CTLA4)humanized mice model,and to provide an effective mouse model for CTLA4-targeted cancer therapeutic antibody research,development,and screening.Methods The animal models were established by CRISPR/Cas9 and then analyzed by polymerase chain reaction(PCR),reverse transcription-PCR(RT-PCR),western blotting,hematoxylin-eosin(HE)staining and fluorescence-activated cell sorting.The mouse melanoma cell line(B16)was injected subcutaneously into CTLA4 humanized mice to observe the anti-tumor effects of intraperitoneal injection of the CTLA4 monoclonal antibody,ipilimumab.Results CTLA4-humanized mice could stably express human CTLA4 but not murine CTLA4.CTLA4-humanized mice survived normally within 6 months after birth,and there were no obvious abnormalities in histopathology and the immune system.In CTLA4-humanized mice,ipilimumab slowed tumor growth.CTLA4-knockout mice did not express CTLA4 and died of autoimmune diseases within 3~5 weeks after birth.Conclusions CTLA4-humanized and CTLA4-knockout mice were established.CTLA4-humanized mice can be used as an animal model for therapeutic antibodyand CTLA4 gene-related drug experiments.