Trp53基因剔除小鼠的制备及其在药物致癌性评价中的应用  

Generation of Trp53 Gene Knockout Mouse and Its Application in Carcinogenicity Assessment of Pharmaceuticals

作　　者：林丹 郑晋华 庄华 石吉晶 林玥琳 王铸钢 匡颖 

机构地区：[1]上海南方模式生物研究中心,上海201203

出　　处：《实验动物与比较医学》2013年第6期418-424,共7页Laboratory Animal and Comparative Medicine

基　　金：上海市科委项目资助（11DZ2292400）,“十二五”科技支撑计划重大专项（2011BAI15802）

摘　　要：目的建立Trp53基因剔除小鼠模型。方法本研究拟通过基因工程技术建立Trp53基因剔除小鼠模型，通过RT—PCR、Western—blot等技术检测野生型、杂合子、纯合子小鼠中Trp53基因的表达；观察三种基因型小鼠自发肿瘤情况并进行病理分析；以N．甲基亚硝基脲（MNU）为致癌剂，探索该模型用于药物致癌性评价的可行性。结果DNA、RNA及蛋白水平的研究证实，已成功获得Trp53基因剔除小鼠模型。初步的表型分析显示：Trp53基因纯合缺失小鼠自出生13周起陆续发生肿瘤，至36周时所有观察纯合小鼠均死于肿瘤，以恶性胸腺淋巴瘤为主（60％）。与野生型小鼠相比，基因杂合缺失小鼠对致癌剂的敏感性显著提升，杂合小鼠经75mg／kg体重的N一甲基亚硝基脲（MNU）诱导，90％的小鼠发生肿瘤，其中86％为恶性胸腺淋巴瘤，而野生小鼠经药物诱导后只有60％发生肿瘤。腹腔注射枸橼酸缓冲液的80只对照组小鼠均未观察到肿瘤的发生。结论这一模型的建立为Trp53基因功能研究和药物致癌性评价提供了理想的动物模型。Objective To establish Trp53 gene knockout mouse. Methods Using ET cloning, homologous recombination, microinjection technology, the Trp53 gene knockout mouse with the deletion of exons 2 to 11 was established. PCR, RT-PCR and Western blot were used to detect the expression of Trp53 in Trp53-/-, Trp53-/- and Trp53-/- mice. The phenotype of the knockout mice was analyzed by spontaneous tumors observation and histopathological examination. The susceptibility of Trp53 het- erozygote （＋/-） and wild type （＋/＋） mice to N-methyl-N-nitrosourea （MNU） was investigated to assess the application of the knockout mouse in carcinogenicity assessment of pharmaceuticals. Results PCR, RT-PCR and Western blot confirmed the deletion of Trp53 gene in the knockout mouse. Trp53/ mice appear normal but are highly susceptible to genesis of a variety of tumors at a very early age-13 weeks of age and all have developed tumor and died by 36 weeks of age. These mice developed many deferent types of tumors, but the most frequently observed tumor is malignant lymphoma, occurring in more than 60% of the mice. Mortality from N-methyl-N-nitrosourea （MNU）-induced lymphomas was much greeter in Trp53＋/- group. After a single intraperitoneal injection of MNU at dose of 75mg/kg in twenty male and female Trp53＋/- and Trp53＋/＋ mice, eighteen male Trp53＋/-mice or female mice developed tumors, espe- cially the malignant lymphoma of thymus, Trp53＋/- in mice 86%. Twenty Trp53＋/ mice and Trp53＋/＋mice per sex receiving a single intraperitoneal injection of citrate buffer appeared normal and no tumor was found. Conclusion The Trp53 gene knockout mice have been established, which can be use in the function study of Trp53 gene and the carcinogenicity assessment of pharmaceuticals.

关 键 词：Trp53基因 基因剔除 肿瘤发生 药物致癌性评价[申图 

分 类 号：R965.1[医药卫生—药理学]