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Medicina

慢性结肠炎相关结肠直肠癌的肿瘤切除术模型，通过卡卡贴片的精确定位

Published: August 24, 2019
doi:
10.3791/59921
, , , , , , , , ,
1Department of General Surgery, The First Affiliated Hospital,Xi’an Jiaotong University, 2Institute of Medical Research,Northwestern Polytechnical University, 3School of Life Sciences,Northwestern Polytechnical University, 4Department of Medicine,Xi’an Jiaotong University, 5Princess Margaret Cancer Center,University of Health Network, 6Department of Pathology,Nanjing Medical University

Summary

提出的协议描述了在小鼠中插入附录（caecal贴片）的方便手术去除，然后是炎症性肠病相关结肠直肠癌的诱导。这种鼠尾切除术模型能够研究附录在人类胃肠道疾病发病机制中的生物学作用。

Abstract

人类附录最近被牵连到在各种复杂疾病的发病机制中起着重要的生物学作用，如结肠直肠癌、炎症性肠病和帕金森病。为了研究附录的功能，建立了肠道疾病相关鼠尾切除术模型，并描述了其分步方案。本报告介绍了小鼠的caecal贴片去除的易制方案，然后使用硫酸盐钠（DSS）和气氧甲烷（AOM）的组合，对慢性结肠炎相关结肠直肠癌进行化学诱导。与假组小鼠相比，在雄性C57BL/6小鼠去除caecal贴片后，IgA特异性细胞和IgA浓度显著降低。同时施用2%的DSS和AOM，导致近80%的小鼠在假和切除组存活，没有显著的体重损失。组织学结果证实结肠炎和不同程度的腺癌。该模型可用于研究附录在维持肠道微生物群落平衡和肠道结肠炎和恶性肿瘤的发病机制中的作用，以及药物靶向疗法的潜在开发。

Introduction

临床切除术是一个标准的外科手术，涉及切除主要由于炎症（例如，附录炎）1，2，3的附录。然而，生物形态人类附录的生物功能仍然有争议的4，5，6。附录已被视为从大肠的cecum中残留的残余物。直到最近，进化、免疫学、形态学和微生物学研究表明，附录可能具有不同的功能。这些作用包括生产免疫球蛋白（例如IgA和IgG），对肠道相关淋巴组织（GALTs）内的适应性免疫反应至关重要的各种B细胞和T细胞，以及用共生微生物补充大肠6,7,8,9,10,11,12.

对先前切除或急性尾炎患者的临床流行病学研究也揭示了其在人类疾病发病机制中的潜在作用，如炎症性肠病（IBD）、结肠直肠癌和非胃肠道疾病疾病（如帕金森病和心血管疾病）13，14，15，16，17，18 。例如，最近一项由75，979名切除术患者组成的大型亚洲人群队列研究表明，结肠直肠癌的切除术与随后的发展之间存在显著联系，而结肠直肠癌是最常见的恶性肿瘤之一，发病率较高，死亡率14，19.因此，建立一个类似于人类的合适的动物切除模型将有助于研究疾病发病机制中附录的生物功能和分子机制。

许多哺乳动物拥有附录或类似附录的器官，包括灵长类动物、拉戈形态动物（如兔子）、一些啮齿动物和20只鼠。对于小型和常用的实验动物，兔子拥有与人类21、22相似的生物形态附录，但兔子中的GALT与人类相比非常大，因为大多数淋巴组织也发现在佩耶的补丁位于小肠和大肠21。此外，兔子表现出不同的淋巴卵泡结构，T细胞分布，和免疫球蛋白密度从人类，这使得研究他们的附录不合适21。

老鼠是研究人类病理生理学和测试各种现存和新颖的植物模型，研究人类病理生理学，研究23、24、25。单白色大淋巴簇在小鼠的caecum的顶点，被称为caecal补丁，被认为执行类似于人类附录26，27，28的功能。然而，在小鼠中，很难将caecal补丁与小鼠的caecum分离开来。到目前为止，在小鼠模型中诱导尾炎的常见外科手术涉及一个相对较大的切口（例如，1⁄2厘米）通过腹壁，以进入整个腹腔（补充表1）29。 30，31，32，33，34，35，36。

本文旨在生成与胃肠道疾病相关的切除模型，本报告提出了小鼠切除cacal斑块的实用手术方案。随后，联合给基因毒性剂AOM和亲炎剂DSS，用于诱导结肠炎相关结肠直肠癌，类似于在人类身上。IBD已被证明是肠癌的危险因素37，38。AOM/DSS诱发慢性结肠直肠癌的组合已经确立，读者可以参考Neufert等人和Thaker等人进行详细程序39，40。这种可重复的快速鼠尾切除术模型可用于研究附录调节的肠炎症和结肠微生物群，特别是在IBD和结肠直肠癌的发展和进展中。

Protocol

所有动物程序均经西安交通大学动物护理使用委员会批准（No.XJTULAC2019-1023）。 1. 小鼠切除术 在手术前1周内，在经认证的无特定病原体（SPF）环境中，将8-10周老的C57BL/6雄性小鼠。 准备以下无菌手术器械：一对微剪刀，一对微钳，两种大小（4-0和8-0）的灭菌性非吸收缝合线，带针架的电凝固笔，75%医用酒精，碘基磨砂（）。例如，碘），和一包无菌棉签。 …

Representative Results

鼠尾切除术模型的建立 这种慢性结肠炎相关结肠直肠癌的切除模型可以通过遵循连续的手术和诱导步骤生成，如图1所示。卡库姆最常见的位置是左和右的iliac fossa后跟腹部的中间线（图2）。使用触觉法在腹部切口前预定位的成功率接近70%（补充信息，表S2）。切除程序主要包括六个重要步骤（<strong class="x…

Discussion

利用手术步骤，在小鼠中获得与结肠炎相关的结肠直肠癌的鼠尾切除术模型。在大多数情况下，由于腹壁位于腹壁下（补充表1，补充表2和图2），如果没有腹腔切除术，很难预先判断其位置。在手术方案中，引入了一个容易接触凸起的步骤，并给出了对cecum位置的定量评估，作为提高cecum预定位精度的指导（图2）。利用解剖特征去除肠的意…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作部分得到中央大学基础研究基金（G2018KY0302）、大学基础研究发展基金（KT00062）、中国国家自然科学基金（81870380）和临床研究奖的支持。西安交通大学第一附属医院XJTU1AF-CRF-2015-029）。作者感谢史成新博士在鼠尾细胞切除模型早期探索阶段提出的技术建议，以及病理学家刘希博士对结肠炎和结肠直肠肿瘤的H&E染色结果的评价。Y.L.进行了手术演示，做了数据分析，并撰写了手稿草稿;J.L.、G.L.、Z.P.和Y.M.参与了外科准备、组织收集以及视频制作;M.Z. 执行流式细胞学和 ELISA;Q.W.和H.X.为生成临床相关鼠种模型提供了技术支持;R.X.Z.设计研究，监督研究，编写和证明手稿;J.S.审阅了手稿。

Materials

Azoxymethane（AOM Sigma-Aldrich,Inc. A5486
Dextran Sulfate Sodium Salt（DSS MP Biomedicals,Inc. 160110
Entoiodine Shanghai likon high technology disinfection co. LTD 310102
digital caliper Ningbo yuanneng trading co. LTD 4859263
4-0 Silk Sutures Yuanlikang co. LTD 20172650032
8-0 Prolene Sutures Yuanlikang co. LTD 20172650032
Electric coagulation pen Chuang mei medical equipment co. LTD 28221777292
disposable syringe 1ml Shengguang medical products co. LTD 3262-2014
disposable syringe 10ml Shengguang medical products co. LTD 3262-2014
75% Medicinal alcohol Shandong anjie high-tech disinfection technology co. LTD 371402AAJ008
Pentobarbital sodium salt Sigma-Aldrich,Inc. 57-33-0
Physiological Saline Shandong qidu pharmaceutical co. LTD H37020766
Absorbent Cotton Swab Henan ruike medical co., LTD RK051
Surgical Instruments-Ophthalmic Jinzhong Shanghai co.LTD WA3050
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Tags

Murine AppendectomyCaecal PatchChronic ColitisColorectal CancerGastrointestinal DiseaseAppendix FunctionSurgical ProcedureMouse ModelCaecumAbdomen IncisionMesenteric Blood VesselsCaecal StumpSuture

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Li, Y., Liu, J., Liu, G., Pan, Z., Zhang, M., Ma, Y., Wei, Q., Xia, H., Zhang, R. X., She, J. Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch. J. Vis. Exp. (150), e59921, doi:10.3791/59921 (2019).
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