结直肠癌（CRC）的癌组织局部免疫状态与CRC的发生、发展和转移关系密切，但缺乏合适的动物模型制约了CRC局部免疫研究。因此构建一个能模拟人体CRC散发性癌灶的，其肿瘤的发生、发展和转移过程可全程无损伤性活体监测的，并方便反复取材的，能同时满足CRC原发灶与转移灶局部免疫状态检测的模型将促进CRC的局部免疫研究进展。本研究中我们将应用小鼠结肠镜，在小鼠体内特异性突变远端结肠内1-2处的结肠隐窝功能干细胞，并同时标记被突变的干细胞，在免疫健全的小鼠体内构建一个满足上述条件的CRC模型，并且在该模型中通过置入结肠内支架的方法缓解因结肠肿瘤生长迅速导致的肠梗阻对小鼠生命和研究周期的影响。构建模型过程不影响机体的免疫功能，且被突变干细胞的病理变化遵循腺瘤-腺癌-转移的发展规律。所构建的模型不仅可用于CRC免疫学研究，同时在CRC的发生、发展研究中也具有广阔的应用前景。

The local immunity in colorectal cancer （CRC）has a close relationship with the generation, development and metastasis of CRC. However, the lack of appropriate animal models highly constrains the investigation of local immunity in CRC. To establish a animal model with following features will promote the research of local immunity in CRC: (i) the model develops sporadic tumors in the colon, which is similar to the human CRC; (ii) the whole process of the generation, development and metastasis of tumor can be monitored in vivo without damage; (iii) the model should have a convenient and repeatable biopsies of colon lesions in living mice during the follow up period of an experiment; (iv) it can meet the requirement of investigation of local immunity both in primary and metastases lesions of CRC. In this project, we will use the murine colonoscopy system to limit mutation and simultaneously mark 1 or 2 functional stem cells of the colonic basal crypt, in the most distal colon for the establishment of a novel orthotopic mouse model of CRC in the immunogenic mice. In the model, we will utilize a metallic stent placed in the colon to relieve the intestinal obstruction, which resulted from rapid tumor growth, and to eliminate the effect to the mice life and research period. Not only the process of the model establishment has no effect to immune function, but also pathological changes of the mutated stem cells present along the entire adenoma-carcinoma-metastasis axis. We believe this model will not only introduce a new powerful tool for the in-vivo study of immunology in CRC, but also has potential application prospects in the research of the generation and development of CRC.

随着分子生物学的发展，国内外学者已通过多种方式构建了结直肠癌（colorectal cancer，CRC）小鼠模型并取得了明显进展，如通过基因工程行相关基因修饰诱导小鼠成瘤，然而这些小鼠模型中，或对小鼠机体损伤较大，影响小鼠正常免疫功能，或小鼠癌变后生存期较短，结直肠癌转移率较低，且大多数呈现肠道多发瘤倾向。截至目前，可模拟人结直肠癌散发性癌灶的免疫功能健全的小鼠模型仍较罕见，限制了结直肠癌发生发展转移机制研究及肿瘤局部免疫研究的进展。有研究显示Smad4与Kras基因的表达可促进结直肠腺癌的发生，而Apc基因的失活可促进腺瘤的形成，因此本研究构建、鉴定并筛选出了Smad4loxP/loxP、ApcloxP/loxP、KrasLSL-G12D/-共三种转基因小鼠，旨在分别通过诱发这三种基因突变来构建小鼠结直肠癌模型，为了探索多基因联合修饰在结直肠癌模型构建中的应用，本项目还构建了Smad4loxP/loxP+Apc loxP/loxP、Smad4loxP/loxP+KrasLSL-G12D/-及ApcloxP/loxP+KrasLSL-G12D/-共三种品系小鼠。同时通过构建可表达Cre重组酶、Luciferase荧光素酶及EGFP绿色荧光蛋白的慢病毒系统LentivirusCre+EGFP+Luciferase，并应用小鼠结肠镜行小鼠远端结直肠粘膜下注射特异性突变1-2处隐窝功能干细胞，成功构建了模拟人CRC散发性癌灶的免疫功能健全的小鼠模型，PCR证实肿瘤的发生源于结肠干细胞基因突变，组织病检证实肿瘤组织的病理变化遵循腺瘤-腺癌-转移的过程，各组成瘤情况比较显示多基因联合修饰相对于单基因修饰具有更高的肿瘤发生率、癌变率及转移率。本研究所构建的模型鼠不仅可通过小鼠结肠镜实时观察肠腔内肿瘤的生长变化情况，还可行活体取材病检，并根据小鼠状态适时置入结肠内支架，有效缓解了肠梗阻并延长了模型鼠的研究周期。由于结肠干细胞被诱变的同时标记了Luciferase荧光素酶基因，因此造模后可通过小鼠活体成像全程无损伤性在体监测肿瘤的发生发展转移情况。综上，该模型鼠肿瘤的发生发展过程与人类散发性结直肠癌类似，且免疫功能健全，因此，不仅可满足结直肠癌发生发展转移机制的研究，还为肿瘤的局部免疫学研究提供了平台。