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

doi:10.13481/j.1671-587x.20200131

摘要/Abstract

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3 d，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-Prkdc^scid IL2rg^tm1Wjl/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9 d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30 d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。

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

Abstract: 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-5F) containing human IL-6, IL-3, IL-15, SCF, and GM-CSF genes was constructed.The 293T cells 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-Prkdc^scid IL2rg^tm1Wjl/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-5F and super-PB. On the 1st, 4th, 5th and 9th 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 d after 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 d after transfection of PB-5F plasmid, 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); at 60 d after transfection, the levels of IL-6, IL-15 and GM-CSF in serum of the mice in stable transfection group were also found. Conclusion: The long-term expression of the PB (piggyBac) transposon system is verified in vitro, and the PB transposon system expressing human IL-6, IL-3, IL-15, SCF and GM-CSF genes is successfully constructed.An immunodeficient mouse model with long-term and stable expressions of human IL-6,IL-15,and GM-CSF is established.

Key words: humanized mouse of immune system, interleukin-6, interleukin-3, interleukin-15, stem cell factor, granulocyte-macrophage cstimulating factor, transposon

中图分类号:

R-332

吕亚楠, 范伟, 胡正. 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[J]. 吉林大学学报(医学版), 2020, 46(01): 176-181.

LYU Yanan, FAN Wei, HU Zheng. Construction of immunodeficient mouse models with long-term expression of human cytokines based on piggyBac transposon system[J]. Journal of Jilin University(Medicine Edition), 2020, 46(01): 176-181.

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