Name: murine model of metastatic liver tumors in the setting of ischemia reperfusion injury
Uploaded: 2019-08-30T13:00:00
Description: Watch this Scientific Journal Video about Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury at JoVE.com

作者感谢萨拉·米内迈尔和亚历山大·科默奇对语言的修正。

所有动物协议均获得机构动物护理和使用委员会的批准，并遵守国家卫生研究院 （NIH） 指南。用于任何外科手术的仪器都经过彻底消毒。
1. 初步准备
<ol><li>在将癌细胞注射到小鼠脾脏之前，高压灭菌和消毒所有在手术过程中使用的仪器。</li>
	<li>消毒和/或高压灭菌加热垫、手术手套、纱布、剪刀对、小钳子、血管扩张器、手术钳和针架。</li>
	<li>准备术后镇痛药（0.1毫克/?...

<ol>
	<li>Riihim&#228;ki, M., Hemminki, A., Sundquist, J., Hemminki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+metastasis+in+colon+and+rectal+cancer.">Patterns of metastasis in colon and rectal cancer.</a> Scientific Reports. 6 (1), 29765 (2016).</li><li>Oki, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+treatment+for+colorectal+liver+metastasis.">Recent advances in treatment for colorectal liver metastasis.</a> Annals of gastroenterological surgery. 2 (3), 167-175 (2018).</li><li>Wolpin, B. M., Mayer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+treatment+of+colorectal+cancer.">Systemic treatment of colorectal cancer.</a> Gastroenterology. 134 (5), 1296-1310 (2008).</li><li>van Golen, R. F., Reiniers, M. J., Olthof, P. B., van Gulik, T. M., Heger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation+in+hepatic+ischemia/reperfusion+injury:+present+concepts+and+potential+therapeutics.">Sterile inflammation in hepatic ischemia/reperfusion injury: present concepts and potential therapeutics.</a> Journal of Gastroenterology and Hepatology. 28 (3), 394-400 (2013).</li><li>Demicheli, R., Retsky, M. W., Hrushesky, W. J. M., Baum, M., Gukas, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+surgery+on+tumor+growth:+a+century+of+investigations.">The effects of surgery on tumor growth: a century of investigations.</a> Annals of Oncology. 19 (11), 1821-1828 (2008).</li><li>Tohme, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+Promote+the+Development+and+Progression+of+Liver+Metastases+after+Surgical+Stress.">Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.</a> Cancer Research. 76 (6), 1367-1380 (2016).</li><li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+model+of+colorectal+cancer.">Orthotopic mouse model of colorectal cancer.</a> Journal of Visualized Experiments. (10), 484 (2007).</li><li>Elkin, M., Vlodavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tail+vein+assay+of+cancer+metastasis.">Tail vein assay of cancer metastasis.</a> Current Protocols in Cell Biology. 19 (19.2), (2001).</li><li>Thalheimer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraportal+injection+model:+A+practical+animal+model+for+hepatic+metastases+and+tumor+cell+dissemination+in+human+colon+cancer.">The intraportal injection model: A practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer.</a> BMC Cancer. 9 (1), 29 (2009).</li><li>Frampas, E., Maurel, C., Thedrez, P., Le Sa&#235;c, P. R. e. m. a. u. d. -., Faivre-Chauvet, A., Barbet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraportal+injection+model+for+liver+metastasis:+Advantages+of+associated+bioluminescence+to+assess+tumor+growth+and+influences+on+tumor+uptake+of+radiolabeled+anti-carcinoembryonic+antigen+antibody.">The intraportal injection model for liver metastasis: Advantages of associated bioluminescence to assess tumor growth and influences on tumor uptake of radiolabeled anti-carcinoembryonic antigen antibody.</a> Nuclear Medicine Communications. 32 (2), 147-154 (2011).</li><li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer.">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Limani, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+portal+vein+injection+for+the+design+of+syngeneic+models+of+liver+malignancy.">Selective portal vein injection for the design of syngeneic models of liver malignancy.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 310 (9), G682-G688 (2016).</li></ol>

本手稿中描述的动物模型基于两种主要方法。第一是识别癌细胞在肝叶中局部化和增殖的能力。二是研究肝缺血再灌注损伤对肿瘤生长和转移的影响。该模型允许在免疫能力小鼠没有继发转移的情况下对肝脏转移进行相关研究。该模型有助于解决转移效率问题，如细胞存活外溢和增殖。
在第一个模型中，首先注射癌细胞，允许微转移性疾病形成。随后，5天后进行肝I/R。该模?...

肝脏是转移性疾病发展的最常见部位之一。死亡率几乎无一例外地归因于与肝脏肿瘤生长相关的并发症。在肝脏转移性实体肿瘤患者中，手术仍然是疾病控制和可能的治疗方法的关键干预措施。然而，绝大多数患者最终都存在复发性疾病，主要在肝脏2，3。在肝手术中，术中出血很常见，经常需要输血和控制出血的不同技术方法，包括血管夹紧方法。然而，这种措施导致肝缺血/再灌注（I/R）到肝脏组织。I/R对肝细胞功能的不利影响已有详细记载。肝脏I/R侮辱通过炎症途径4恢复血流期间点燃炎症级联。肝I/R损伤不仅导致肝功能衰竭，而且目前的证据还表明，I/R损伤刺激肿瘤细胞粘附，促进转移形成发生率和现有微转移性疾病5的生长。我们以前曾报道过，手术应激诱导免疫细胞的活化，这不仅有助于原发性肿瘤的生长，而且通过捕获循环6中的癌细胞，促进转移。
在这里，我们详细描述了建立肝转移小鼠肿瘤模型的技术。在此模型中，我们还提出了一种诱导肝缺血再灌注损伤的方法，该方法在肝切除术期间临床上对临床上存在的手术应激作用。癌症注射和肝I/R的结合方法可以成功地解释原发性肿瘤切除患者CRLM的发展。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin</td>
			<td>Fisher scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Stretomysin</td>
			<td>Fisher scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>SH3023701</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Hyclone</td>
			<td>sh30042.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Flask 75cm</td>
			<td>5 Cells Star</td>
			<td>658170</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml PP Conical Tubes</td>
			<td>BioExcell</td>
			<td>41021037</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Giibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisherbrand</td>
			<td>1376152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cautry</td>
			<td>Bovie</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular clamp</td>
			<td>Finescience tools</td>
			<td>18055-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Serrefine clamp applicator with lock</td>
			<td>Fine science toosl</td>
			<td>FST-18056-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissor</td>
			<td>Fine science toosl</td>
			<td>FST-15021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Dilator</td>
			<td>Fine science toosl</td>
			<td>FST-00276-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic fixator Retraction system</td>
			<td>Fine science toosl</td>
			<td>FST-18200020</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine science toosl</td>
			<td>FST-11019-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine science toosl</td>
			<td>FST-11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 polypropylene suture</td>
			<td>Ethicon</td>
			<td>K881H</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Harvard Apparatus</td>
			<td>72-8826</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Fisher scientific</td>
			<td>1443915</td>
			<td></td>
		</tr>
		<tr>
			<td>Clipper</td>
			<td>Oster</td>
			<td>559A</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine solution</td>
			<td>Medline</td>
			<td>MDS093945</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml 25G</td>
			<td>BD safety Glide</td>
			<td>305903</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe 0.5 ml</td>
			<td>BD insulin Syringes</td>
			<td>32946</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton -Tipped Applicator</td>
			<td>Fisher Scientific</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>Leica</td>
			<td>LR92240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoplasma Elisa Kit</td>
			<td>Roche</td>
			<td>11663925910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Putney</td>
			<td>#056344</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>NADA</td>
			<td>#139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>ALT strip</td>
			<td>Heska</td>
			<td>15809554</td>
			<td></td>
		</tr>
		<tr>
			<td>AST strip</td>
			<td>Heska</td>
			<td>15809542</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH strip</td>
			<td>Heska</td>
			<td>15809607</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine model of metastatic liver tumors in the setting of ischemia reperfusion injury

肝缺血和再灌注（I/R）损伤是一种常见的临床挑战，仍然是一个不可避免的病理生理学过程，已被证明诱导多个组织和器官损伤。尽管最近取得了进步和治疗方法，但总体发病率仍然不能令人满意，特别是在有潜在宫腔异常的患者中。在恶性肿瘤生长和转移的背景下，手术I/R被怀疑是调节肿瘤复发的促进剂。本文旨在描述一种临床相关的肝I/R和结肠直肠肝转移模型。在此过程中，我们旨在协助其他研究者建立和完善这一模型，为他们的日常研究实践，以更好地了解肝I/R对促进肝转移的影响。

使用上述方案，所有野生型（C57BL6）小鼠（n = 20）均接受肝脏转移模型。所有注射的小鼠有或没有缺血性再灌注损伤，直到牺牲日期。癌症注射肝脏的示意图图图图 1A说明了导致部分肝缺血（70%） 的入口三重体（肝动脉、入口静脉和胆管）的夹紧侮辱对中位数和左侧叶。在缺血再灌注损伤后2-3周内，可观察到肝转移次数的增加。注射MC38癌细胞的小鼠被随机分为假和I/R组。<s...

Watch this Scientific Journal Video about Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury at JoVE.com

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

详细描述了临床相关的结肠直肠癌肝转移（CRLM）肿瘤模型以及肝缺血再灌注（I/R）对肿瘤生长和转移的影响。该模型可以帮助更好地了解手术诱导的肝脏转移生长促进机制。

缺血再灌注损伤中转移性肝肿瘤的模样

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

实验和临床证据表明，肝缺血再灌注可以通过循环癌细胞和微米静病的生长增加转移性病的形成。这种可重复的穆林模型允许对转移的建立和肝脏内预先存在的肿瘤的生长进行相关研究。肝脏手术、出血和败血症休克都与肝缺血再灌注有关，可影响转移性疾病的生长。这些技术允许研究这种现象，以努力更好地诊断和防止缺血再灌注对肿瘤生长在临床环境中的有害影响。在确认在麻醉的8至12周男性C57黑色6小鼠对头趾捏没有反应后，用波维酮碘磨砂对裸露的腹壁皮肤进行消毒，并使用牙齿钳子抬起皮肤。使用手术手术刀，在皮肤上做一个中线切口，并抬起腹部肌肉和腹腔，以方便创建一个三厘米的中线切口，以暴露腹部内容物。在切口两侧和西腓工艺下放置一个 hemostat 后，向下拉并胶带将尾巴向下延伸，并使用六英寸无菌棉尖施用器将脾脏从胰腺脂肪组织中分离和暴露。接下来，旋涡癌细胞悬浮的兴趣，分离任何细胞团，并加载细胞到0.5毫升胰岛素注射器配备了28规格的针头。慢慢地小心地将100微升细胞注入脾脏的尖端，将针刺边的棉尖放在一边，吸收任何可能的肿瘤溢出到腹膜腔中，同时在注射过程中增加温和的压力以稳定脾脏。成功的注射将明显通过肝脏颜色的变化。当所有细胞都交付后，将一块PBS浸泡的无菌纱布放在解剖区域，将鼠标放在加热垫上15分钟，使癌细胞在系统中循环。要诱发缺血和再灌注损伤，请使用两个湿润的棉尖轻轻地将肠从腔中移出，以暴露入口静脉，将鼠标置于解剖显微镜下。提起中位，左向肝叶对隔膜。在中叶和右侧叶之间放置一个小的湿润棉签，以创造足够的空间进行夹紧，并小心地将容器扩张器钳子穿过入口三合会，并使用容器扩张器钳子，使用 10 厘米的 4-0 聚丙烯缝合线通过后侧，并抬起入口三合会。使用带锁的微丝神经夹施用器，将微血管夹放在左侧和中位肝叶上，以遮挡入口三合会中的所有结构。观察发白时，取出4-0聚丙烯缝合线和小棉签，小心地将肠子返回到腹腔。用一块新的 PBS 浸泡纱布盖住腹壁，用塑料包装盖住纱布，以尽量减少蒸发损失。然后将鼠标放回加热垫上 60 分钟。在整个缺血间隔内，想象右中叶、左中叶和横向叶的苍白发白，以确认缺血损伤的证据。在受伤期结束时，拆下夹具。要进行脾脏切除术，请小心地用光滑的钳子抬起脾脏，并使用手持式烧灼装置烧灼脾脏血管，以避免过度出血。在烧灼部分的血管transtrans去除脾脏，并立即使用4-0聚丙烯缝合线，以双层缝合图案关闭肌肉和皮肤切口。然后将鼠标放回其家庭笼子，并进行监控，直到完全恢复。手术应激显著增加肝脏内预先建立的微肉量。与非缺血重灌小鼠相比，即使在缺血再灌注两周后，也观察到这种差异。肝缺血再灌注和肿瘤细胞注射可受室温和细胞生存能力的影响，因此请小心控制这些变量。该技术有助于研究手术应激和肝损伤对转移性疾病生长的影响。这将有助于研究人员更好地了解手术引起的肿瘤生长现象。

Research

JoVE Journal

Cancer Research

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

肝大肠癌转移的高级动物模型：成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...