P53^+/-基因敲除小鼠对尿烷致癌性验证试验的敏感性研究

目的：通过尿烷多次给药初步验证自主建立的P53^+/-基因敲除小鼠（B6-Trp^tm1/NIFDC）模型是否比野生型C57BL/6小鼠对尿烷敏感，为国内用于药物临床前安全评价致癌实验短期体内试验提供可用的基因修饰动物模型。方法：在C57BL/6来源的ES细胞中，通过打靶技术敲除p53基因的第2~5外显子后对获得的P53^+/-基因敲除小鼠进行PCR基因型鉴定，再与普通C57BL/6小鼠杂交，获得的后代再通过PCR筛选获得基因敲除动物（命名为B6-Trp^tm1/NIFDC）。尿烷验证试验共设计3个组，阴性对照组（野生型小鼠）：给予生理盐水，尿烷组1（P53^+/-敲除小鼠）：给予尿烷，尿烷组2（野生型小鼠）：给予尿烷，给药方式为腹腔注射共给药3次，给药剂量为1 000 mg·kg^-1。给药后第8周开始肿瘤触诊观察，每周1次。给药后6个月后进行计划剖解，通过大体剖检和组织病理学检查以评价P53^+/-敲除小鼠对尿烷致癌作用的敏感性。结果：成功获得杂合的P53^+/-基因敲除小鼠，给予尿烷后可诱发P53^+/-敲除小鼠发生肝脏血管扩张、肺腺瘤、颌下腺血管瘤及脾脏淋巴瘤，病变发生率分别为94.4%、33.3%、5.6%、5.6%；野生型小鼠肝脏血管扩张及肺脏腺瘤的发生率分别为36.8%、10.5%；阴性对照组无肿瘤发生。结论：本研究初步验证自主建立的P53^+/-基因敲除小鼠对尿烷的致癌敏感性高于野生型小鼠，该模型有望将来是临床前药物安全性评价致癌性实验短期体内试验候选模型之一。

Objective: To validate whether P53^+/- gene knockout mice model, constructed by ourselves and named as B6-Trp53 tm1/NIFDC is more sensitive to carcinogenic urethane than wild type C57BL/6 mice by multiple dosing of urethane in order to provide an animal model for short-term carcinogenicity study of preclinical safety evaluation of drugs in China. Methods: The genotypes of P53^+/- knockout mice were identified by PCR after knockouting exons 2-5 of p53 gene by gene targeting in ES cells on a C57BL/6 background, and then hybridized with normal C57BL/6 mice. The obtained offspring(named B6-Trp^tm1/NIFDC)were then screened by PCR to obtain p53 gene knockout animals. There were three different groups, negative control: wild type C57BL/6 mice treated with saline solution;urethane group 1: P53^+/- knockout mice treated with urethane and urethane group 2: wild type C57BL/6 mice treated with urethane. 1 000 mg·kg^-1 urethane as a carcinogenic agent were administrated 3 times by the route of intraperitoneal injection. The weekly tumor palpation began 8 weeks after the administration of urethane. All animals were necropsied after 6 months of dosing. Dying and dead animals were necropsied as soon as possible. The sensitivity of P53^+/- knockout mice to the carcinogenesis of urethane was assessed through gross necropsy and histopathology examination. Results: The knockout mice with heterozygous p53 gene was successfully obtained. Urethane could induce liver angiectasis, lung adenoma, submandibular gland hemangioma and spleen lymphoma in P53^+/- knockout mice and the incidence of the above pathological changes were 94.4%、33.3%、5.6%、5.6%, respectively. The incidence of liver angiectasis and lung adenoma in wild type C57BL/6 mice treated with urethane were 36.85%, 10.5%, respectively. No tumor was observed in negative control group. Conclusion: The present study preliminarily validated that P53^+/- knockout mice model established by ourselves was more sensitive to urethane than that of the wild type C57BL/6 mice and might be a promising candidate short-term carcinogenicity testing system for preclinical safety evaluation of drugs in the future.