文章摘要
张 苗,周 霞,王 敏,孙可帅,马硕怡,郑小红,王敬博,韩 英.肝细胞Lamp-2a时间特异性基因敲除大鼠的构建及评估[J].,2019,19(7):1206-1211
肝细胞Lamp-2a时间特异性基因敲除大鼠的构建及评估
Establishment and Evaluation of Time-conditionally Hepatocyte-specific Lamp-2a Gene Knockout Rats
投稿时间:2018-10-13  修订日期:2018-11-09
DOI:10.13241/j.cnki.pmb.2019.07.002
中文关键词: 他莫昔芬  Lamp-2a  Cre/loxp系统  时间特异性敲除
英文关键词: Tamoxifen  Lamp-2a  Cre/loxp  Time Conditionally Knockout
基金项目:国家自然科学基金面上项目(81770569;81870398);国际合作与交流项目(81820108005)
作者单位E-mail
张 苗 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032 zhmfmmu@163.com 
周 霞 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032  
王 敏 上海市儿童医院 上海 200062  
孙可帅 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032  
马硕怡 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032  
郑小红 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032  
王敬博 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032  
韩 英 空军军医大学西京消化病医院肿瘤生物学国家重点实验室 陕西 西安 710032  
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中文摘要:
      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-2aloxp/-大鼠和Alb-CreERT2工具鼠杂交繁殖,得到基因型Lamp-2aloxp/-Alb-CreERT2+大鼠;后者再与Lamp-2aloxp/loxp杂交得到双臂loxp阳性和Alb-CreERT2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。
英文摘要:
      ABSTRACT Objective: To establish and evaluate a time-conditionally hepatocyte-specific Lamp-2a gene knockout rat model (L2AKO) 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-2a 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 ex- pression of Lamp-2a gene in hepatocytes, while the expression of Lamp 2a gene in kidney was normal as wild type rats (WT). Western and qPCR results showed that the protein and mRNA level of Lamp-2a in liver decreased significantly. As for the body weight, there was no obvious difference between L2AKO and WT rats. However, the biochemical test of liver function suggested that the AST level of L2AKO rats was higher than WT rats. Conclusion: The Cre/loxp system and tamoxifen induction method have been used to successfully establish a time-conditionally hepatocyte-specific Lamp-2a gene knockout rat model. And the growth of L2AKO rats was not affected in this process. Therefore, it could be a better animal model for further studies of bile acid metabolism.
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