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hKDR
人源化
遗传
修饰
小鼠
模型
建立
刘甦苏
DOI:10.12300/j.issn.1674-5817.2022.154实验动物与比较医学 Laboratory Animal and Comparative MedicineApr.2023,43(2)范昌发,博士,研究员,博士生导师,中国食品药品检定研究院模式动物研究室主任,组建了该院的遗传修饰动物模型制作平台,构建了全球首个人源化新冠小鼠模型hACE2-KI,自主建立了致癌模型KI.C57-ras V2.0等代表性小鼠模型,并广泛用于我国新冠疫苗研发及新药临床前致癌性评价。发表论文100余篇,包括Science(2篇)、Nature、Cell Host&Microbe、STTT(3篇)、NSR、EMI(3篇)、Cancer Science、Cell Research、Virus(2篇)、Vaccine(4篇)等杂志;获得Cell Press杂志社评选的“2020年度中国区最受欢迎论文”;申请专利13项,授权10项;承担或组织申请国自然基金、国家重大新药创制专项、国家传染病重大专项、国家病原学专项课题等17项;荣获中国药学会科学技术奖二等奖1项,北京市科学技术进步奖一等奖1项、二等奖1项。自主研制遗传修饰大、小鼠模型50余种。研究工作被英国The Economist、美国Cell Press、中国科学网、北京科技报等媒体专题报道。被科技部评选为“全国科技系统抗击新冠肺炎疫情先进个人”。刘甦苏,毕业于中国农业大学,动物医学专业硕士,副主任技师,现工作于中国食品药品检定研究院,一直从事与药品检定和安全评价相关的遗传修饰模式动物技术工作,涉及医药学术领域中的肿瘤类动物模型、传染病动物模型、人源化动物模型三大类研究范围。工作期间主持、参与国家级及院级课题共6项,发表期刊论文共29篇,其中第一作者7篇(SCI论文1篇,核心期刊论文6篇);合编专著1部;专利申请/授权共10项,其中专利授权4项,新申请专利6 项;起草并发布中国实验动物学会团体标准2项。获北京实验动物行业协会科学技术二等奖、2020年北京实验动物行业协会先进个人称号。hKDR+/+人源化及Rag1-/-基因缺陷新型双靶点遗传修饰荷瘤小鼠模型的建立刘甦苏,吴勇,曹愿,赵皓阳,翟世杰,孙晓炜,李琳丽,范昌发(中国食品药品检定研究院实验动物资源研究所,国家啮齿类实验动物资源库,北京 102629)摘要 目的通过增强血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)人源化小鼠(hKDR+/+)接纳不同来源的小鼠肿瘤细胞系的潜力,建立能支持多种肿瘤细胞系成瘤的、可评价针对VEGFR靶点药物的新型荷瘤小鼠模型。方法首先建立基于hKDR+/+人源化小鼠模型评价针对VEGFR靶点抗体体内活性的方法,同时采用CRISPR/Cas9技术构建重组激活基因1(recombination activating gene 1,Rag1)敲除的C57BL/6N小鼠(命名为Rag1-/-小鼠)。然后将Rag1-/-小鼠与hKDR+/+小鼠交配,筛选获得双基因纯合、双靶点基因修饰的hKDR+/+/Rag1-/-小鼠。最后在hKDR+/+、Rag1-/-、hKDR+/+/Rag1-/-和C57BL/6N小鼠上测试不同小鼠品系来源肿瘤细胞系的体内肿瘤生长差异。结果针对VEGFR靶点设计的抗体在hKDR+/+小鼠体内表现出明显的抗肿瘤活性,与PBS组相比,肿瘤体积明显减小(P0.01),肿瘤质量明显减轻(P0.05)。Rag1-/-小鼠、hKDR+/+/Rag1-/-小鼠的外周血B细胞和T细胞数量均明显减少(P0.05,P0.001)。C57BL/6小鼠来源的MC38细胞接种7 d后,在hKDR+/+/Rag1-/-、Rag1-/-、C57BL/6N和hKDR+/+四组小鼠体内均可见肿瘤生长。BALB/c小鼠来源的CT26细胞接种10 d后,仅在hKDR+/+/Rag1-/-和Rag1-/-小鼠体内见到肿瘤生长,在 C57BL/6N 及hKDR+/+小鼠中均未见肿瘤生长;接种后 3 周,hKDR+/+/Rag1-/-小鼠的成瘤体积明显大于Rag1-/-小鼠(P0.01)。结论获得了Rag1基因敲除小鼠,并进一步筛选获得新型hKDR+/+/Rag1-/-双靶点基因修饰小鼠模型;Rag1基因缺失时,不同品系小鼠来源的肿瘤细胞系更易成瘤。这提示可以通过降低hKDR+/+人源化小鼠的免疫反应能力,增强或扩大该模型小鼠接纳肿瘤细胞系的范围,构建多种可用于评价VEGFR靶点药物的荷瘤小鼠模型。关键词 hKDR+/+人源化小鼠;Rag1-/-基因敲除小鼠;VEGFR靶点;抗体;肿瘤中图分类号 Q95-33;R-332 文献标志码 A 文章编号 1674-5817(2023)02-0103-09 人类疾病动物模型 Animal Models of Human Diseases基金项目 国家科技重大专项课题“创新生物技术药评价及标准化关键技术研究”(2018ZX09101-001)第一作者 刘甦苏(1981),女,硕士,副主任技师,主要从事模式动物研究。E-mail:通信作者 范昌发(1970),男,研究员,主要从事模式动物研究。E-mail:。ORCID:0000-0001-5556-2025103实验动物与比较医学 Laboratory Animal and Comparative MedicineApr.2023,43(2)Establishment of hKDR+/+Humanized and Rag1-/-Gene Knockout Double Genetically Modified Mouse ModelLIU Susu,WU Yong,CAO Yuan,ZHAO Haoyang,ZHAI Shijie,SUN Xiaowei,LI Linli,FAN Changfa(Institute for Laboratory Animal Resources,National Institutes for Food and Drug Control;National Rodent Laboratory Animal Resources Center,Beijing 102629,China)Correspondence to:FAN Changfa(ORCID:0000-0001-5556-2025),E-mail:ABSTRACT ObjectiveThrough improving the potential of vascular endothelial growth factor receptor(VEGFR)-humanized mouse model(hKDR+/+)with C57BL/6N background to allow the growth of different mouse tumor cell lines,to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR.Methods Firstly,a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR+/+humanized mouse model was established.Recombinant activating gene 1(Rag1)knockout mice(Rag1-/-)were established using CRISPR/Cas9 technology.Then these Rag1-/-mice were crossed with hKDR+/+mice to get a double gene modified homozygous hKDR+/+/Rag1-/-mouse model by screening.Finally,tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth.ResultsAntibodies designed for VEGFR showed significant anti-tumor activity in hKDR+/+mice,which significantly reduced tumor volume and weight compared with the PBS group(P0.01,P0.05).The number of B cells and T cells in the peripheral blood of Rag1-/-mice and hKDR+/+/Rag1-/-mice decreased(P0.05,P0.001).Tumors were observed in hKDR+/+/Rag1-/-,Rag1-/-,wild-type,and hKDR+/+mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice.Tumors were only observed in groups of hKDR+/+/Rag1-/-and Rag1-/-mice,but not in the wild-type and hKDR+/+mice after 10 d of inoculation with CT26 cells derived from BALB/c mice.After 3 weeks of inoculation,the tumor volume of hKDR+/+/Rag1-/-mice was significantly larger than that of Rag1-/-mice(P0.01).ConclusionRag1 knockout mice were obtained and a novel hKDR+/+/Rag1-/-double genes modified mouse model was further screened.The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency.The results suggest that the reduced immune response of hKDR+/+humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model.As a result,more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.Key words hKDR+/+humanized mice;Rag1-/-knockout mice;VEGFR target;Antibodies;Tumor肿瘤血管生成与肿瘤生长、侵入和转移关系密切,因此