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

作　　者：白家驷 毛青 BAI Jiasi;MAO Qing(Institute of Infectious Diseases,Chongqing Key Laboratory of Infectious Diseases,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]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,重庆400038

出　　处：《陆军军医大学学报》2023年第13期1405-1412,共8页Journal of Army Medical University

摘　　要：目的构建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。Objective 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 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 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 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：CRISPR/Cas9技术 Rosa26基因位点 人源化 嵌合肝 转基因小鼠 尿激酶纤溶酶原激活物 

分 类 号：R-332[医药卫生] R322.47R394-3