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应用CRISPR/Cas9技术构建Tet-on调控表达uPA的人源化肝细胞嵌合小鼠
Construction of humanized chimeric mice with Tet-on regulating liver specific expression of uPA using CRISPR/Cas9 technology
- 陆军军医大学学报 2023年45卷第13期 页码：1405-1412
- 作者机构：
400038 重庆，陆军军医大学(第三军医大学)第一附属医院传染病研究所，重庆市传染病研究重点实验室
- 作者简介：
[ 毛青，E-mail: qingmao@tmmu.edu.cn MAO Qing, E-mail: qingmao@tmmu.edu.cn
- 基金信息：
DOI：10.16016/j.2097-0927.202303025
中图分类号： R-332;R322.47;R394-3收稿：2023-03-06，
修回：2023-04-12，
纸质出版：2023-07-15
- 稿件说明：
移动端阅览
白家驷, 毛青. 应用CRISPR/Cas9技术构建Tet-on调控表达uPA的人源化肝细胞嵌合小鼠[J]. 陆军军医大学学报, 2023,45(13):1405-1412.
Jiasi BAI, Qing MAO. Construction of humanized chimeric mice with Tet-on regulating liver specific expression of uPA using CRISPR/Cas9 technology[J]. Journal of Army Medical University, 2023, 45(13): 1405-1412.
白家驷, 毛青. 应用CRISPR/Cas9技术构建Tet-on调控表达uPA的人源化肝细胞嵌合小鼠[J]. 陆军军医大学学报, 2023,45(13):1405-1412. DOI： 10.16016/j.2097-0927.202303025.
Jiasi BAI, Qing MAO. Construction of humanized chimeric mice with Tet-on regulating liver specific expression of uPA using CRISPR/Cas9 technology[J]. Journal of Army Medical University, 2023, 45(13): 1405-1412. DOI： 10.16016/j.2097-0927.202303025.
摘要
目的
2
构建Tet-on调控小鼠肝脏特异性表达uPA基因的NOD-SCID背景的转基因小鼠
并通过人肝癌细胞移植修复小鼠亚急性肝损伤进而构建人源化肝细胞嵌合小鼠。
方法
2
采用CRISPR/Cas9技术，在gRNA引导下，Cas9核酸内切酶于Rosa26基因位点切割并编辑
插入目的基因pTight-uPA-pA-Alb promoter-rtTA-pA表达框。使目的基因能跟随染色体稳定遗传，生物学功能保持稳定。新培育的转基因小鼠分为4组：诱导组(
n
=17)、移植组(
n
=16)、未诱导组(
n
=5)和空白对照组(
n
=5)。诱导组经强力霉素诱导3周，肝脏中表达的uPA能造成小鼠肝脏亚急性损伤；再通过人肝癌细胞移植修复。移植4周后，通过小鼠血清生化学、组织病理学，评估该人源化肝细胞嵌合小鼠的效果。
结果
2
诱导组小鼠血清中uPA、丙氨酸氨基转移酶(ALT)均高于未诱导组和空白对照组(
P
＜0.01)；移植组人白蛋白含量显著高于未移植组(
P
＜0.01)。组织病理学HE染色发现人肝癌细胞参与小鼠肝组织的修复，激光共聚焦显微镜观察显示鼠肝脏中有人肝癌细胞聚集并表达人白蛋白，组化染色人角质蛋白(CK18)表达成阳性。
结论
2
新构建的转基因小鼠能通过强力霉素诱导小鼠肝脏中uPA表达，造成肝脏亚急性损伤，并通过人源肝癌细胞移植修复进而构建人源化肝细胞嵌合小鼠。
Abstract
Objective
2
To construct NOD-SCID transgenic mice with Tet-on regulated liver specific expression of urokinase plasmin activator (uPA)
repair subacute liver injury in mice through human liver cancer cell transplantation
and thereby establish humanized liver cell chimeric mice.
Methods
2
With CRISPR/Cas9 technology
under the guidance of gRNA
Cas9 endonuclease was cut and edited at Rosa26 locus
and then inserted into the target gene pTight-uPA-pA-Alb promoter rtTA-pA expression frame in order to enable the target gene to follow the stable inheritance of chromosomes and maintain stable biological functions. The newly cultivated transgenic mice were divided into induction group (
n
=17)
transplantation group (
n
=16)
non-induction group (
n
=5)
and blank control group (
n
=5). The induction group was induced by doxycycline for 3 weeks
and then uPA expression in the liver caused subacute liver injury in mice
which was repaired through transplantation of human liver cancer cells. After 4 weeks of transplantation
the effect of humanized liver cell chimeric mice was evaluated through mouse serum biochemistry and histopathology.
Results
2
The serum levels of uPA and alanine aminotransferase (ALT) were significantly higher in the induction group than those the non-induction group and blank control group (
P
<
0.01)
so was the human albumin content in the transplantation group than that of the 2 groups without transplantation (
P
<
0.01). HE staining showed that human hepatoma cells participated in the repair of mouse liver tissues. Laser confocal microscopy displayed that human hepatoma cells gathered in the mouse liver and expressed human albumin. Histochemical staining showed that human keratin (CK18) was positive in the mouse liver tissues.
Conclusion
2
The newly constructed transgenic mice can induce uPA expression in the mouse liver through doxycycline
causing subacute liver damage
and then construct humanized chimeric mice through transplantation and repair of human liver cancer cells.
关键词
Keywords
references
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