SMAD同源物4抑癌基因在小鼠散发性结直肠癌模型中的作用  

Role of SMAD family member 4 tumor-suppressor gene in sporadic colorectal tumorigenesis of transgenic mouse model

作　　者：黎华丽 程煌荣 杨超[1] 黄朔阳 郑勇斌[1] Li Huali;Cheng Huangrong;Yang Chao;Huang Shuoyang;Zheng Yongbin(Department of Gastointestinal Surgery,Renmin Hospital of Wuhan University,Wuhan 430064,China)

机构地区：[1]武汉大学人民医院胃肠外科,430064

出　　处：《中华实验外科杂志》2020年第11期2071-2074,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81372553)。

摘　　要：目的构建Apc^loxP/loxP+SMAD同源物4(Smad4)^loxP/loxP双转基因小鼠模拟人散发性结直肠癌,观察Smad4在结直肠癌发生发展中的作用。方法分别将Apctm1Tyj/J小鼠、Smad4tm2.1Cxd/J小鼠与C57BL/6J小鼠(均购自美国杰克逊实验室)杂交和回交转换遗传背景形成杂合子小鼠C57BL/6-ApcloxP/-/J(记为ApcloxP/-)、C57BL/6-Smad4loxP/-(记为Smad4loxP/-),将ApcloxP/-和Smad4loxP/-小鼠杂交建系并通过聚合酶链反应(PCR)筛选出Apc^loxP/loxP+Smad4loxP/loxP小鼠。通过小鼠结肠镜分别向Apc^loxP/loxP+Smad4loxP/loxP小鼠和C57BL/6J小鼠(各12只)结直肠黏膜下注射前期构建好慢病毒LentivirusCre-IRES-Luciferase,腹腔注射荧光素酶D(D-luciferase)后在小动物活体成像系统(IVIS)下成像并通过结肠镜动态观察成瘤情况。最后对小鼠瘤灶取材行苏木精-伊红(HE)染色观察其病理改变。组间比较采用t检验。结果成功培育出Apc^loxP/loxP+Smad4loxP/loxP双转基因小鼠22只。在LentivirusCre-IRES-Luciferase诱导下发生突变并形成散发性结直肠肿瘤病灶,经病理组织学证实为腺癌,可通过IVIS系统及小鼠结肠镜动态观察其情况,至12周末成瘤率33%(4/12),C57BL/6J小鼠未观察到瘤变。两组体重分别为(22.58±1.21)、(21.36±1.01)g,比较差异无统计学意义(t=0.892,P>0.05);日龄分别为(63±1)、(62±1)d,差异无统计学意义(t=0.121,P>0.05)。结论本研究构建的Apc^loxP/loxP+Smad4loxP/loxP双转基因小鼠结肠组织在LentivirusCre-IRES-Luciferase诱导下发生癌变,成功模拟人散发性结直肠癌的发生过程,证实Smad4抑癌基因的条件性敲除可促进结直肠癌的发生。Objective To construct Apc^loxP/loxP+Smad4^loxP/loxP double transgenic mouse model which can imitate the process of tumoregenisis of human sporadic colorectal cancer(CRC)and to verify the important role of SMAD family member 4(Smad4)gene.Methods Thegenotypic milieuof adenomatous polyposis coli gene(Apc)tm1Tyj/J and Smad4tm2.1Cxd/Jmicewas transformed into C57BL/6J mice(all mouse were purchased from The Jackson Laboratory)and mice were then crossed to establish atransgenic mouse strain.Finally,offspring mice with Apc^loxP/loxP+Smad4^loxP/loxP were generated by polymerase chain reaction(PCR).Transgenic mice and C57BL/6J mice(12,respectively)were injected with LentivirusCre-IRES-Luciferase into the intestinal mucosa under colonoscope.After intraperitoneal injection of D-luciferase,the mice were imaged through in vivo imaging system(IVIS)to observe tumor progression dynamically.And the tumor tissues were sampled and stained with Hematoxylin-Ehong(HE)to verify the tumoregenicity of mice.The t test was used to compare the two groups.Results 22 doubletransgenic mice with Apc^loxP/loxP+Smad4^loxP/loxP were successfully bred,tissue neoplasia wereinduced by LentivirusCre-IRES-Luciferase and formed sporadic colorectal tumor lesions which were confirmed by IVIS and histology.At the end of 12 weekend,tumoregenesis rate was 33%(4/12),but tumor lesion was not observed in all C57BL/6J mice.The body weight of the experimental and control group were(22.58±1.21),(21.36±1.01)g,respectively,with no statistically significant difference(t=0.892,P>0.05);and the age of mice were(63±1),(62±1)d,respectively,with no statistically significant difference(t=0.121,P>0.05).Conclusion The transgenic mice with Apc^loxP/loxP+Smad4^loxP/loxP constructed in this experiment induced tumoregenesis by LentivirusCre-IRES-Luciferase,successfully simulated the process of human sporadic CRC,and confirmed that the knockout of Smad4 gene can promote the occurrence of CRC.

关 键 词：结直肠癌 APC基因 SMAD4同源物4 模型 动物 

分 类 号：R735.34[医药卫生—肿瘤] R-332[医药卫生—临床医学]