摘要
目的:构建肝细胞Lamp-2a时间特异性基因敲除大鼠模型(L2AKO)并对其评估,为后续胆汁酸代谢研究提供研究基础。方法:将引进的Lamp-2^(aloxp/-)大鼠和Alb-Cre^(ERT2)工具鼠杂交繁殖,得到基因型Lamp-2a^(loxp/-)Alb-Cre^(ERT2+)大鼠;后者再与Lamp-2a^(loxp/loxp)杂交得到双臂loxp阳性和Alb-Cre^(ERT2)阳性的大鼠,于4-6周时经他莫昔芬的诱导实现对肝细胞Lamp-2a基因的时间特异性敲除。分别通过qPCR方法、Western-Blot技术、免疫组织化学方法对Lamp-2a的敲除结果进行验证。结果:免疫组织化学结果显示:该模型可选择性敲除肝细胞Lamp-2a,而肾脏Lamp-2a正常表达;Western和qPCR结果显示肝脏Lamp-2a的水平明显降低。L2AKO与WT大鼠相比体重变化无明显差别。肝功生化检测发现L2AKO大鼠AST明显高于WT组大鼠。结论:采用Cre/loxp系统及他莫昔芬诱导的方法成功构建肝细胞Lamp-2a时间特异性基因敲除大鼠,并且L2AKO大鼠生长未受影响,为胆汁酸代谢研究建立了较好的动物模型。
Objective:To establish and evaluate a time-conditionally hepatocyte-specific Lamp-2 a gene knockout rat model(L2 AKO)and to provide an opportunity for further studies of bile acid metabolism.Methods:First,Lamp-2aloxp/-rats and Alb-CreERT2 rats were interbred to obtain Lamp-2aloxp/-Alb-CreERT2+rats,which were then interbred with Lamp-2aloxp/loxp rats,After that,we obtained the double-arm loxp positive and Alb-CreERT2 positive rats.Finally,time-conditionally hepatocyte-specific Lamp-2 a gene knockout rats can be acquired by tamoxifen induction during 4-6 weeks.The model was verified by qPCR,Western-Blot and immunohistochemistry.We also evaluate the weight and liver serum function of this model.Results:Immunohistochemical results showed that there was almost no expression of Lamp-2 a gene in hepatocytes,while the expression of Lamp 2 a gene in kidney was normal as wild type rats(WT).Western and q PCR results showed that the protein and m RNA level of Lamp-2 a in liver decreased significantly.As for the body weight,there was no obvious difference between L2 AKO and WT rats.However,the biochemical test of liver function suggested that the AST level of L2 AKO rats was higher than WT rats.Conclusions:The Cre/loxp system and tamoxifen induction method have been used to successfully establish a time-conditionally hepatocyte-specific Lamp-2 a gene knockout rat model.And the growth of L2 AKO rats was not affected in this process.Therefore,it could be a better animal model for further studies of bile acid metabolism.
作者
张苗
周霞
王敏
孙可帅
马硕怡
郑小红
王敬博
韩英
ZHANG Miao;ZHOU Xia;WANG Min;SUN Ke-shuai;MA Shuo-yi;ZHENG Xiao-hong;WANG Jing-bo;HAN Ying(State Key Laboratory of Cancer Biology,Xijing Hospital of Digestive Diseases,Air Force Medical University,Xi'an,Shaanxi,710032,China;Children's Hospital of Shanghai,Shanghai,200062,China)
出处
《现代生物医学进展》
CAS
2019年第7期1206-1211,共6页
Progress in Modern Biomedicine
基金
国家自然科学基金面上项目(81770569
81870398)
国际合作与交流项目(81820108005)
