摘要
目的构建能模拟人散发性结直肠癌发生发展的ApcloxP/loxP+ KrasLSL-Gl2D/-双转基因小鼠模型.方法将C57 BL/6-Apctm1Tyj/J小鼠(ApcloxP)、B6.129S4-Krastm4Tyj/J(KrasLSL-G12D)小鼠与C57 BL/6小鼠转换遗传背景后再杂交建系,通过聚合酶链反应(PCR)和荧光定量PCR鉴定小鼠及其子代基因型,筛选出基因型为Apclox/loxP+ KrasLSL-G12D/-的双转基因小鼠.将小鼠分为两组C57BL/6J(10只)和ApcloxP/loxP+ KrasLSL-G12D/-(10只),在小鼠结肠镜下向小鼠肠黏膜内注射表达Cre重组酶和Luciferase的慢病毒,通过小动物活体成像系统(IVIS)动态观察小鼠成瘤情况,对小鼠瘤变组织取材进行苏木精-伊红(HE)染色观察其组织病理学变化情况,两组比较采用t检验.结果本研究成功培育出双转基因ApcloxP/loxP+ KrasLSL-G12D/-小鼠品系共24只,实验组与对照组小鼠日龄分别为(62±2)、(63±2)d,差异无统计学意义(t=0.343,P> 0.05);两组小鼠体重分别为(21.39±1.40)、(22.35±1.37)g,差异无统计学意义(t=0.682,P>0.05).向小鼠肠黏膜内注射慢病毒后,至12周末经IVIS观察及组织病理学证实,ApcloxP/loxP+KrasLSL-G12D/-组10只中有4只小鼠结直肠发生突变而形成肿瘤病灶,成瘤率40%,而C57BL/6J组小鼠未发生肿瘤.结论本研究成功通过表达Cre酶的慢病毒诱导ApcloxP/loxP+KrasLSL-G12D/-双转基因模型小鼠结直肠发生癌变,较好地模拟了人散发性结直肠癌的发生发展过程.
Objective To construct ApcloxP/loxP+ KrasLSL-G12D/- double transgenic mouse model that can simulate the occurrence and development of human sporadic colorectal cancer. Methods The Apctm1Tyj/J (ApcloxP) and B6.129S4-Krastm4Tyj/J (KrasLSL-G12D) mice were purchased from Jackson Laboratory, and their genotypic milieu were transformed into C57BL/6J mice and then crossed to establish a transgenic mouse strain. The genotypes of mice were discerned by polymerase chain reaction (PCR) and fluorescence quantitative PCR. Finally, double transgenic mice with ApcloxP/loxP+ KrasLSL-G12D/- genotype were bred. Mice were divided into two groups: C57BL/6J mice group (n=10) and ApcloxP/loxP+ KrasLSL-G12D/- mice group (n=10). Lentivirus which can express Cre recombinant enzyme and Luciferase, were injected into the intestinal mucosa of mice under colonoscopy (FB-8V, Japan PENTAX company). The tumorigenesis of the mice can be observed dynamically by in vivo Imaging System (IVIS). The tumor tissues of the mice were sampled and stained with Hematoxylin-Ehong (HE) to verify the tumorigenicity of the mice. The difference of the two groups was compared with t test. Results 24 ApcloxP/loxP+ KrasLSL-G12D/- mice were successfully bred. The age of mice in the experimental group and control group was (62±2) days and (63±2) days, respectively, with no statistically significant difference (t=0.343, P>0.05). The body mass of the two groups was (21.39±1.40) g and (22.35±1.37) g, respectively, with no statistically significant difference (t=0.682, P>0.05). Lentivirus was injected into the intestinal mucosa of mice shaping colorectal cancer. At the end of 12 weekend, 4 of 10 mice (tumorigenesis rate were 40%) in ApcloxP/loxP+ KrasLSL-G12D/- group were found burden colorectal adenocarcinoma confirmed by IVIS and histology. However, tumor lesion was not observed in all C57BL/6J mice. Conclusion In this study, the colorectal carcinogenesis of ApcloxP/loxP+ KrasLSL-G12D/- transgenic mice were induced by Lentivirus expressing Cre enzyme, which successfully imitated the germination and development of human sporadic colorectal cancer.
作者
黄晓东
郑勇斌
杨玉杰
杨超
黎华丽
程煌荣
Huang Xiaodong;Zheng Yongbin;Yang Yujie;Yang Chao;Li Huali;Cheng Huangrong(Department of Gastrointestinal Surgery,Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第10期1791-1794,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(81372553).
