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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 determine whether the independently established model of P53 + (+/-) knockout mice (B6-Trp ~ (tm1) / NIFDC) is more active than urethane Sensitive, for the domestic use in clinical preclinical safety evaluation of carcinogenic short-term in vivo experiments to provide available genetically modified animal model. Methods: PCR-based genotyping of P53 +/- mouse knockouts in C57BL / 6-derived ES cells was performed by knocking out the second to fifth exons of p53 gene by targeting After hybridization with normal C57BL / 6 mice, the obtained offspring were knocked out by PCR screening (named as B6-Trp ~ (tm1) / NIFDC). In the urethane validation test, three groups were designed: Negative control group (wild type mice): saline, urethane group 1 (P53 +/- (+/-) knockout mice): urethane and urethane group 2 (Wild-type mice): given urethane, administered by intraperitoneal injection co-administered 3 times the dose of 1 000 mg · kg -1. Tumor palpation was started 8 weeks after the administration, once a week. Six months after dosing, a planned dissection was conducted to assess the sensitivity of P53 +/- (+/-) knockout mice to carcinogenicity of urethane by gross and histopathological examination. Results: Heterozygous P53 +/- mice were successfully knocked out and the hepatic vasodilation, pulmonary adenoma and submandibular gland hemangioma were induced by urethane in mice with P53 +/- knockout Spleen lymphoma and pathological changes were 94.4%, 33.3%, 5.6% and 5.6% respectively. The incidence of hepatic vasodilation and lung adenoma in wild-type mice were 36.8% and 10.5% respectively. There was no tumor in the negative control group. CONCLUSIONS: This study preliminarily validates that the independently established P53 +/- mouse has higher carcinogenic sensitivity to urethane than wild-type mice and is expected to be a preclinical drug safety assessment for carcinogenicity in the future. Short-term One of the candidate models for in vivo testing.