Name: dual bioluminescence imaging of tumor progression and angiogenesis
Uploaded: 2019-08-01T13:00:00
Description: Watch this Scientific Journal Video about Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis at JoVE.com

本研究得到国家重点研发项目(2017YFA0103200)、国家自然科学基金(81671734)和天津市科技支撑项目重点项目(18YFZCSY00010)的支持。中央大学 (63191155)。我们感谢格洛丽亚·南斯的修订,这些修订对于提高我们手稿的质量很有价值。

实验必须符合关于将动物用于研究的国家和体制条例。必须获得进行实验的权限。动物的处理和研究的实验程序遵循南开大学动物护理和使用委员会准则,该指南符合国家卫生研究院(NIH)批准的《动物护理指南》。
1. 吕吕路-RFP (RR) 和 LV-Rluc-RFP-HSV-ttk (RRT) 慢病毒包装和生产
注:pLV-RR在启动子EF1+下携带雷尼拉荧光酶(Rluc)和红色荧光蛋白(RFP)的基因序列,而pLV-RRT携?...

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+therapeutic+implications.">Tumor angiogenesis: therapeutic implications.</a> The New England Journal of Medicine. 285, 1182-1186 (1971).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis.">Tumor angiogenesis.</a> The New England Journal of Medicine. 358, 2039-2049 (2008).</li><li>Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+enigma+of+multicolor+bioluminescence+of+firefly+luciferase.">Molecular enigma of multicolor bioluminescence of firefly luciferase.</a> Cellular and Molecular Life Sciences. 68, 1167-1182 (2011).</li><li>Nakatsu, T., 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=Structural+basis+for+the+spectral+difference+in+luciferase+bioluminescence.">Structural basis for the spectral difference in luciferase bioluminescence.</a> Nature. 440, 372-376 (2006).</li><li>McMillin, D. W., 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=Tumor+cell-specific+bioluminescence+platform+to+identify+stroma-induced+changes+to+anticancer+drug+activity.">Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity.</a> Nature Medicine. 16, 483-489 (2010).</li><li>Madero-Visbal, R. A., Hernandez, I. C., Myers, J. N., Baker, C. H., Shellenberger, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+bioluminescent+imaging+of+xenograft+progression+in+an+orthotopic+mouse+model+of+HNSCC.">In situ bioluminescent imaging of xenograft progression in an orthotopic mouse model of HNSCC.</a> Journal of Clinical Oncology. 26, 17006 (2008).</li><li>Wang, R., 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=Molecular+Imaging+of+Tumor+Angiogenesis+and+Therapeutic+Effects+with+Dual+Bioluminescence.">Molecular Imaging of Tumor Angiogenesis and Therapeutic Effects with Dual Bioluminescence.</a> Current Pharmaceutical Biotechnology. 18, 422-428 (2017).</li><li>Rivera, L. B., Cancer Bergers, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis,+from+foe+to+friend.">Tumor angiogenesis, from foe to friend.</a> Science. 349, 694-695 (2015).</li><li>Zhang, K., 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=Enhanced+therapeutic+effects+of+mesenchymal+stem+cell-derived+exosomes+with+an+injectable+hydrogel+for+hindlimb+ischemia+treatment.">Enhanced therapeutic effects of mesenchymal stem cell-derived exosomes with an injectable hydrogel for hindlimb ischemia treatment.</a> ACS Applied Materials &amp; Interfaces. 10, 30081-30091 (2018).</li><li>Du, W., 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=Enhanced+proangiogenic+potential+of+mesenchymal+stem+cell-derived+exosomes+stimulated+by+a+nitric+oxide+releasing+polymer.">Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.</a> Biomaterials. , 70-81 (2017).</li><li>Lee, 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=Autocrine+VEGF+signaling+is+required+for+vascular+homeostasis.">Autocrine VEGF signaling is required for vascular homeostasis.</a> Cell. 130, 691-703 (2007).</li><li>Dewhirst, M. W., Cao, Y., Moeller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cycling+hypoxia+and+free+radicals+regulate+angiogenesis+and+radiotherapy+response.">Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response.</a> Nature Reviews. Cancer. 8, 425-437 (2008).</li><li>Wigerup, C., Pahlman, S., Bexell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+hypoxia+and+hypoxia-inducible+factors+in+cancer.">Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer.</a> Pharmacology &amp; Therapeutics. 164, 152-169 (2016).</li><li>Wong, P. 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=Dual-action+combination+therapy+enhances+angiogenesis+while+reducing+tumor+growth+and+spread.">Dual-action combination therapy enhances angiogenesis while reducing tumor growth and spread.</a> Cancer Cell. 27, 123-137 (2015).</li><li>Mezzanotte, L., van &#39;t Root, M., Karatas, H., Goun, E. A., Lowik, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Molecular+Bioluminescence+Imaging:+New+Tools+and+Applications.">In vivo Molecular Bioluminescence Imaging: New Tools and Applications.</a> Trends in Biotechnology. 35, 640-652 (2017).</li><li>Du, W., Tao, H., Zhao, S., He, Z. X., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+applications+of+molecular+imaging+in+cardiovascular+disease+and+stem+cell+therapy.">Translational applications of molecular imaging in cardiovascular disease and stem cell therapy.</a> Biochimie. 116, 43-51 (2015).</li><li>Liu, J., 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=Synthesis,+biodistribution,+and+imaging+of+PEGylated-acetylated+polyamidoamine+dendrimers.">Synthesis, biodistribution, and imaging of PEGylated-acetylated polyamidoamine dendrimers.</a> Journal of Nanoscience and Nanotechnology. 14, 3305-3312 (2014).</li><li>Branchini, B. R., 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=Red-emitting+chimeric+firefly+luciferase+for+in+vivo+imaging+in+low+ATP+cellular+environments.">Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments.</a> Analytical Biochemistry. 534, 36-39 (2017).</li><li>McLatchie, A. 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=Highly+sensitive+in+vivo+imaging+of+Trypanosoma+brucei+expressing+&amp;#34;red-shifted&amp;#34;+luciferase.">Highly sensitive in vivo imaging of Trypanosoma brucei expressing &amp;#34;red-shifted&amp;#34; luciferase.</a> PLoS Neglected Tropical Diseases. 7, e2571 (2013).</li></ol>

在本协议中,描述了一种非侵入性双BLI方法,用于监测肿瘤发育和血管生成。BLI报告器系统是首次开发,包含HSV-ttk/GCV自杀基因,用于跟踪肿瘤进展和通过Rluc成像在体内的回归。同时,通过氟成像利用Vegfr2-Fluc-KI小鼠评估肿瘤血管生成。这种带肿瘤小鼠模型能够提供一个实用的平台,通过双BLI在单一小鼠具有高相关性、可重复性和可翻译性,为连续和非侵入性跟踪肿瘤发育和肿瘤血管生成提供实用平台。...

血管生成是癌症从小,局部肿瘤到更大的,潜在的转移肿瘤1,2进展的一个基本过程。肿瘤生长与血管生成的相关性成为肿瘤学研究领域的重点之一。然而,传统的测量形态变化的方法未能同时监测活体动物的肿瘤进展和血管生成。"
肿瘤细胞的生物发光成像(BLI)是监测肿瘤生长的一种特别合适的实验方法,因为它的非侵入性、敏感性和特异性3、4、5、6.BLI技术基于荧光素酶在排放生物发光时可以催化特定基质氧化的原理。植入的肿瘤细胞中表达的荧光素酶与注射的基质发生反应,这种基质可以通过活的成像系统检测,信号间接反映细胞数或细胞定位在体内6、7的变化。
除了肿瘤生长,肿瘤血管生成(癌症进展的关键步骤)也可以通过BLI技术使用Vegfr2-Fluc-KI转基因小鼠8,9,10可视化。血管内皮生长因子(Vegf)受体2(Vegfr2),一种类型的Vegf受体,主要表达在成年小鼠的血管内皮细胞11。在Vegfr2-Fluc-KI转基因小鼠中,萤火虫荧光素酶(Fluc)的DNA序列被敲入内源性Vegfr2序列的第一个外子。因此,Fluc以与小鼠血管生成水平相同的方式表示(显示为 BLI 信号)。为了生长超过几毫米的大小,肿瘤从现有的血管中招募新的血管,这高度表达由肿瘤细胞1的生长因子触发的Vegfr2。这为使用Vegfr2-Fluc-KI转基因小鼠通过BLI非侵入性监测肿瘤血管生成开辟了可能性。
在此协议中,建立了一个带肿瘤的小鼠模型,分别通过萤火虫荧光素酶(Fluc)和雷尼拉荧光酶(Rluc)成像来监测单个小鼠的肿瘤进展和血管生成(图1)。创建 4T1 细胞系 (4T1-RR),通过 Rluc 成像可稳定增长 Rluc 和红色荧光蛋白 (RFP) 来跟踪细胞生长。为了进一步调查血管生成在肿瘤的进展和回归的动态变化,另一个4T1细胞系(4T1-RRT)被创建,表达自杀基因单纯疱疹病毒截断胸腺激酶(HSV-ttk),Rluc和RFP。通过对甘西洛韦(GCV)的分管,HSV-ttk表达细胞被选择性地消融。基于这些细胞系,建立了Vegfr2-Fluc-KI小鼠的肿瘤承载模型,作为连接肿瘤进展和体内肿瘤血管生成的实验模型。

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin-0.53 mM EDTA</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Tubes</td>
			<td>Axygen Scientific</td>
			<td>MCT-105-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Tubes</td>
			<td>Corning Glass Works</td>
			<td>601052-50</td>
			<td></td>
		</tr>
		<tr>
			<td>293T</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>4T1</td>
			<td>ATCC</td>
			<td>CRL-2539</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Dish</td>
			<td>Corning Glass Works</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Plate</td>
			<td>Corning Glass Works</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Shanghai Lishen Scientific</td>
			<td>Hfsafe-900LC</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidine S Hydrochloride (BSD)</td>
			<td>Sigma-Aldrich</td>
			<td>15205</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8</td>
			<td>MedChem Express</td>
			<td>HY-K0301</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Tegulated Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>4111</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine (CTZ)</td>
			<td>NanoLight Technology</td>
			<td>479474</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin Potassium Salt</td>
			<td>Caliper Life Sciences</td>
			<td>119222</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>BIOIND</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>Ti-E/U/S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ganciclovir (GCV)</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001129</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics Software</td>
			<td>GraphPad Software</td>
			<td>Graphpad Prism 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe Needles</td>
			<td>Becton Dickinson</td>
			<td>328421</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>691477H</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral Packaging System</td>
			<td>Biosettia</td>
			<td>cDNA-pLV03</td>
			<td></td>
		</tr>
		<tr>
			<td>Liposome</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging Software</td>
			<td>Caliper Life Sciences</td>
			<td>Living Imaging Software 4.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>IVIS Lumina II</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Medium</td>
			<td>Invitrogen</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Glass Works</td>
			<td>R21031399</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>H9268-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Gibco</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>SORVALL ST 16R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>Thermo Sorvall ST 16 ST16R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low Temperature Refrigerator</td>
			<td>Haier</td>
			<td>DW-86L338</td>
			<td></td>
		</tr>
		<tr>
			<td>XGI-8 Gas Anesthesia System</td>
			<td>XENOGEN Corporation</td>
			<td>7293</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual bioluminescence imaging of tumor progression and angiogenesis

血管生成作为肿瘤进展的关键环节,已成为抗肿瘤治疗的研究热点和目标。然而,没有可靠的模型来同时以视觉和敏感的方式追踪肿瘤进展和血管生成。生物发光成像具有灵敏度高、特异性强、测量准确等优点,在活成像中具有独特的优势。这里介绍的一个协议,通过注射一个雷尼拉荧光酶标记的鼠乳腺癌细胞系4T1到转基因小鼠与血管生成诱导萤火酶酶表达建立肿瘤小鼠模型。该小鼠模型提供了一个有价值的工具,通过单只小鼠的双生物发光成像,实时监测肿瘤进展和血管生成。该模型可广泛应用于抗肿瘤药物筛选和肿瘤研究。

在本实验中,利用4T1细胞建立了乳腺癌小鼠模型,以研究肿瘤生长与肿瘤血管生成的关系(图1)。首先,包装了两个慢病毒,其基因序列分别表达Rluc/RFP(LV-RR)和Rluc/RFP/HSV-ttk(LV-RRT),如先前报告7。然后,分别通过转导LV-RR和LV-RRT创建两个不同的4T1细胞系,分别命名为4T1-RR和4T1-RRT。经过3天的药物筛选,在荧光显微镜下观察4T1-RR和4T1-RRT,以检测转导效?...

Watch this Scientific Journal Video about Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis at JoVE.com

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

肿瘤进展和血管生成的双生物发光成像

该协议描述了通过双生物发光成像实时监测肿瘤进展和血管生成情况的肿瘤携带小鼠模型。

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

血管生成在肿瘤转移和进展中起着重要作用。在该协议中，我们将使用双生物发光成像方法监测乳腺癌小鼠模型中的动态肿瘤进展和血管生成。在此模型中，我们可以分别通过萤火虫荧光酶和雷尼拉荧光酶成像，在单个小鼠中同时跟踪肿瘤生长和血管发生。该模型可广泛应用于抗肿瘤药物筛选和肿瘤学研究。这种双生物发光模型可用于跟踪肿瘤进展和回归，并检测肿瘤相关的分子过程，以响应治疗策略。血管生成是肿瘤进展的一个关键过程。因此，以视觉和敏感的方式监测肿瘤进展和血管生成对于开发有效的肿瘤治疗是必要的。我们实验室的学生张凯月、王晨和尚晨将演示这些程序。对于扁病毒包装和生产种子1倍10至6的293T细胞在两毫升DMEM，辅以10%胎儿牛血清每井到六井板过夜培养在37摄氏度和5%二氧化碳的加湿培养。第二天早上，将7.5微升脂体与250微升的最小基本介质混合在两个独立的1.5毫升管中，在室温下孵育5分钟。为了准备DNA解决方案，将质粒扁病毒RR载体和帮助质粒添加到250微升的最小基本介质在个别1.5毫升管，如表中概述。在孵化结束时，以滴滴的方式轻轻地将DNARR溶液加入脂质体悬浮液中，并允许DNA在室温下与脂质膜结合20分钟。当混合物孵育时，用一毫升新鲜培养培养物替换293T细胞培养物的上一提液，并轻轻地将0.5毫升的适当脂体DNA混合物添加到每一个井中。将细胞返回细胞培养箱12至16小时，然后用一毫升新鲜培养培养剂补充抗生素，更换每井中的上能。再孵育36小时后，将中继细胞集中到每扁病毒株的锥形管中，通过离心沉淀293T细胞。然后将含有扁病毒RR的超级纳酸转移到单个1.5毫升无菌聚丙烯储存管中，在零下80摄氏度下储存。对于4T1乳腺肿瘤细胞中基因表达的延脱病毒转导，用一毫升新鲜RPMI基细胞培养基，辅以胎儿牛血清和一毫升的扁病毒蛋白，取代六井4T1细胞培养板每个井中的上能。然后在每毫升多聚苯丙胺中加入8微克，轻轻移液几次混合。离心板，以帮助提高转导效率，并返回细胞培养箱4至12小时。在孵育结束时，用补充血清和抗生素的新鲜培养基替换每井中的上经剂，以去除任何扁病毒颗粒和多聚布雷恩。要选择扁病毒转导细胞，以一到三比一比四的转导4T1细胞培养，用选择培养基，每两到三天改变一次培养基。经过七天的选择培养，在荧光倒置相对比显微镜下查看转导细胞培养，并计算红色荧光蛋白阳性或RFP-4T1细胞的数量以及三个视觉领域的所有4T1细胞，以估计RFP阳性比。要建立带肿瘤的小鼠模型，在每60毫米培养板用两毫升胰腺素-EDTA分离细胞之前，用PBS清洗80%的汇合传感器细胞培养物。当细胞从板底提升时，每盘用5至10毫升血清补充介质停止反应。并转移培养物到单独的15毫升离心管计数。将细胞稀释至每100微升培养基的10至6个细胞，无血清浓度。皮下将100微升的4T1-RR细胞系注射到麻醉肿瘤模型小鼠的左肩，将4T1-RRT细胞系的100微升注入同一小鼠的右肩。注射后，将每只小鼠放在适当的恢复区域，并配备热支撑，直到完全恢复。每天对肿瘤质量进行7天的治疗，以确认小鼠具有肿瘤。在植入后的第七天，在实验结束之前，每天向每只含肿瘤小鼠注射50微克，每公斤甘西洛维。对于肿瘤生长，打开活成像系统。初始化活成像软件并初始化系统。当系统初始化时，使用胰岛素注射器将每只小鼠注射到回溯杆中，用适当体积的 coelenterazine 进行成像。将注射小鼠放入相机室，并立即获取小鼠多张照片，以便从 4T1 细胞获取雷尼拉荧光酶信号，直到生物发光信号消失。在雷尼拉荧光素酶成像后10分钟，使用胰岛素注射器将适当体积的D-荧光素注射到每只动物体内。D-荧光素注射10分钟后，将小鼠放入相机室，获取小鼠后体的几幅图像，以便从血管生成获取萤火虫荧光素酶信号。在扁病毒转导后，超过99%的细胞为RFP阳性，两个转导细胞系之间观察到的细胞培养形态没有差异。随后，转导细胞的生物发光成像显示，两个细胞系都发出强度相似的强生物发光信号。将转导细胞系皮下注射到转基因肿瘤携带小鼠模型中后，可在同一动物中出现D-luciferin的情况下，通过萤火虫荧光酶信号评估血管生成引起的肿瘤生长。在植入后7天，Ganciclovir给法导致4T1-RRT细胞死亡，导致这些细胞建立的肿瘤中的雷尼拉荧光素酶信号显著减少。萤火虫荧光酶信号也随着雷尼拉荧光酶表达的增加而增加，在荧光素酶下降后减少。综合起来，这些结果表明，肿瘤血管生成和肿瘤生长之间有直接的关系。事实上，甘西洛维诱导的肿瘤细胞死亡可能导致肿瘤血管生成抑制。此外，作为血管生成标志的抗血管内皮生长因子受体II的免疫保持揭示了4T1-RR肿瘤组织部分比4T1-RRT肿瘤部分更显著建立的微血管结构，这与甘西洛维尔施用后观察到的萤火虫百合酶信号的下降一致。该协议最关键的步骤是使用两种荧光素酶，如FLuc和RLuc同时跟踪细胞和血管生成。该技术可用于治疗不同地点的癌细胞，并可广泛应用于病毒癌模型。这种小鼠模型允许将HSV-ttk/GCV跟踪系统的肿瘤生长和抗肿瘤效应动态地在活的动物中可视化。需要生物安全II级设施来防止扁病毒转染，因为介质中含有可能对人类有害的扁病毒颗粒。

Research

JoVE Journal

Medicine

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

双模式成像，皮肤组织氧合和血管功能

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

科研

医学

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vivo Dual Substrate Bioluminescent Imaging

In vivo Dual Substrate Bioluminescent Imaging

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...