基于piggyBac转座子系统构建长期表达人细胞因子的免疫缺陷小鼠模型  

Construction of immunodeficient mouse models with long-term expression of human cytokines based on piggyBac transposon system

作　　者：吕亚楠 范伟 胡正[1] LYU Yanan;FAN Wei;HU Zheng(Academy of Translational Medicine/Institute of Immunology,First Hospital,Jilin University,Changchun 130021,China)

机构地区：[1]吉林大学第一医院转化医学院/免疫学研究所

出　　处：《吉林大学学报（医学版）》2020年第1期176-181,共6页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81870091,81570145)

摘　　要：目的:探讨表达人白细胞介素6 (IL-6)、白细胞介素3 (IL-3)、白细胞介素15 (IL-15)、干细胞生长因子(SCF)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的piggyBac (PB)转座子系统在免疫缺陷小鼠体内的长效表达情况,为改善人源化小鼠模型中人免疫细胞的重建提供简单长效的新方法。方法:构建含有绿色荧光蛋白(GFP)基因的PB转座子质粒(PB-GFP),构建含有人IL-6、IL-3、IL-15、SCF和GM-CSF基因的PB转座子质粒(PB-5F)。293T细胞分为阴性对照组(未转染质粒)、阳性对照组(转染pLVTHM质粒)、瞬时转染组(转染PB-GFP质粒)和稳定转染组[共同转染PB-GFP和转座酶质粒(super-PB)]。转染后每3d,采用流式细胞术检测各组细胞中GFP阳性(GFP+)细胞百分率。将NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NCG)小鼠分为瞬时转染组和稳定转染组,瞬时转染组小鼠尾静脉高压注射PB-5F质粒,稳定转染组小鼠尾静脉高压注射PB-5F质粒和super-PB质粒。小鼠尾静脉高压注射后1、3、5和9d及随后每周1次采血分离血清,ELISA法检测各组NCG小鼠血清中IL-6、IL-3、IL-15、SCF和GM-CSF水平。结果:PB-GFP转染293T细胞30d后,稳定转染组GFP+细胞百分率(4.61%±0.42%)明显高于阳性对照组(0.58%±0.05%)和瞬时转染组(0.86%±0.10%)(P<0.05)。NCG小鼠尾静脉高压注射PB-5F质粒,注射后第30天,稳定转染组小鼠血清中IL-6、IL-15和GM-CSF水平均高于瞬时转染组(P=0.048,P=0.051,P=0.045);注射后第60天,稳定转染组小鼠血清中仍能检测到IL-6、IL-15和GM-CSF。结论:体外验证了PB转座子系统的长效表达,成功构建了具有人IL-6、IL-3、IL-15、SCF和GM-CSF基因的PB转座子载体的质粒,建立了长效稳定表达人IL-6、IL-15和GM-CSF的免疫缺陷小鼠模型。Objective:To investigate the long-term expression of the piggyback(PB)transposon system expressing human interleukin-6(IL-6),interleukin-3(IL-3),interleukin-15(IL-15),stem cell factor(SCF)and granulocyte-macrophage cstimulating Factor(GM-CSF)genes in the immunodeficient mice,and to provide a simple,long-term and new method for improving the reconstruction of human immune cells in the humanized mouse models.Methods:The PB transposon plasmid(PB-GFP)containing the GFP gene was constructed,and the PB transposon plasmid(PB-5 F)containing human IL-6,IL-3,IL-15,SCF,and GM-CSF genes was constructed.The293 Tcells were divided into negative control group(non-transfection),positive control group(transfected with pLVTHM),transient transfection group transfected with PB-GFP plasmid and stable transfection group[transfected with PB-GFP plasmid together with transposase plasmid(super-PB)].The proportions of GFP+cells in varions groups were measured by flow cytometry every three days after transfection.The NOD.Cg-Prkdcscid IL2 rgtm1 Wjl/SzJ(NCG)mice were divided into transient transfection group and stable transfection group.The mice in transient transfection group were transfected with PB-GFP alone,and the mice in stable transfection group were transfected with PB-5 Fand super-PB.On the 1 st,4 th,5 th and 9 th days and the following every week after the transfection,the blood samples were collected,and the serum was separed;the levels of IL-6,IL-3,IL-15,SCF,and GM-CSF in serum of the mice in various groups were detected by ELISA.Results:At 30 dafter transfection of PB-GFP,the percentage of GFP+cells of the mice in stable transfection group(4.61%±0.42%)was significantly higher than those in positive control group(0.58%±0.05%)and transient transfection group(0.86%±0.10%)(P<0.05).At 30 dafter transfection of PB-5 Fplasmid,the levels of serum IL-6,IL-15 and GM-CSF of the NCG mice in stable transfection group were significantly higher than those in transient transfection group(P<0.05);at60 dafter transfection,the levels of IL-6,I

关 键 词：免疫系统人源化小鼠 白细胞介素6 白细胞介素3 白细胞介素15 干细胞生长因子 粒细胞-巨噬细胞集落刺激因子 转座子 

分 类 号：R-332[医药卫生]