Name: orthotopic injection of breast cancer cells into the mice mammary fat pad
Uploaded: 2019-01-20T13:00:00
Description: Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad at JoVE.com

作者感谢中国国家自然科学基金 (拨款编号: 8187111、81673924、81774039、81873111)、北京自然科学基金 (赠款编号: 772095、716884、7202083) 和北京医院徐霞基金会。中医药 (赠款号, xx201701)。我们感谢中国医学科学院实验研究中心的刘长珍教授提供的生物发光图像。

动物实验是根据中国实验动物管理局立法的规定和一般建议进行的, 并得到了首都医科大学动物护理和使用委员会的批准 (参考编号:)。aeei-00552)。使用6至8周的雌性 balbbe 小鼠。
1. 细胞和动物的制备
<ol><li>手术前一天 , 剃光第四周围的头发 , 露出手术区域。<ol>
<li>用一只手将鼠标紧紧地握在后面 , 以确保鼠标不会自由移动 , 打开鼠标将其腹部暴露给操作员 , 然后用另一只手用...

<ol>
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本研究中使用的细胞为 4t1-luc2, 小鼠三阴性乳腺癌细胞, 具有荧光素酶标记, 是投资药物抗肿瘤和抗肿瘤作用的有用工具,因为它们具有高度侵入性 2,19。.荧光素酶, 稳定到下一代, 被用来表明活的肿瘤细胞, 无论是在乳腺和其他遥远的器官 20.在某些情况下, 肿瘤内缺氧和营养缺乏会导致生物发光信号减少, 原发肿瘤的体积与肿瘤进展后期?...

乳腺癌是全世界女性死亡的主要原因。随着乳腺癌发病率的逐步上升, 乳腺癌已成为对公共卫生的严重挑战1。小鼠癌症模型是临床前和临床研究之间的良好桥梁, 良好的模拟小鼠疾病模型将提高疾病和医学研究的准确性。
原发性肿瘤生长开始了恶性肿瘤的发展, 而转移和并发症是大多数癌症患者死亡和生活质量差的主要原因。几种小鼠模型被用来模拟人类乳腺癌的病理 2,3,4。异种移植模型被广泛用于癌症研究, 以了解病理特征, 筛选药物的安全性和有效性5, 6,7.基因工程小鼠 (gem) 是通过针对某些癌基因或肿瘤抑制基因而产生的, 以模仿人类乳腺癌.创业板具有相对简单和统一的背景, 可以了解基因在癌变进展中的作用;然而, 人工环境和背景有限, 研究转移病理及相关治疗 9.人类癌细胞虽然具有人类病理特征, 但只能植入免疫缺陷小鼠体内, 而肿瘤-宿主免疫相互作用不足可能会导致偏颇的结果10。
固体肿瘤的发生地直接影响到疾病的生物和病理特征 11, 12,13.由于癌变进展是肿瘤细胞、间质细胞、免疫细胞、炎症细胞、生长因子和蛋白酶相互作用的复杂结果,植入原位的原发肿瘤将提供更好的洞察和模仿与化学剂或皮下注射肿瘤细胞引起的肿瘤相比, 癌变过程更准确。用于诱发肿瘤的化学剂可能对研究人员和环境有害, 甚至在一些国家被禁止。由于没有乳腺脂肪垫环境, 皮下注射的病理进展可能与真正的乳腺癌患者不同, 他们的癌症源于乳腺脂肪垫并刺激。皮下注射的缺点鼓励使用原位模型来研究肿瘤的生长。在早期的研究中, 经过7次原位移植后出现的高度转移性 mda-mb-231 肿瘤表明了注射部位14的重要性.最近, 经手术将乳腺癌细胞原位植入乳腺脂肪垫的报道为15,16例。随着乳腺垫环境的出现, 肿瘤的生长和向远处器官的迁移覆盖了与病理相关的部位的乳腺癌的整个过程, 使这一模型成为人类疾病进展的缩影。然而, 手术后, 皮肤会自动尝试自愈, 这可能会带来干扰正常乳腺癌起源的潜在风险, 并影响结果。
我们比较了一些乳腺癌模型, 建立了微创原位模型, 以研究药物对乳腺癌进展的潜在影响17,18.在这项研究中, 提出了一个视频协议如何正声表入乳腺癌细胞在乳房脂肪垫在一个简单的, 侵入性较小的方式。这种不经手术的原位注射方法在许多方面都是有利的。首先, 操作简单快捷, 每只鼠标约1分钟。其次, 原发肿瘤病灶从正确的病理部位开始, 涵盖了乳腺癌从肿瘤生长到其他器官转移的整个肿瘤发生过程, 为研究乳腺癌提供了良好的实验动物模型。肿瘤细胞与肿瘤微环境的相互作用。此外, 估计乳腺癌各阶段的治疗效果也是一个有价值的模型。这种方法的目的是提供一个动物模型, 以最大限度地模仿人类乳腺癌的进展。

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			<td height="48" style="height:49px;width:149px;">anesthesia machine</td>
			<td style="width:235px;">Midmark Corporation, Dayton, OH, USA</td>
			<td style="width:96px;">Matrx VMS</td>
			<td style="width:172px;">anesthesia</td>
		</tr>
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			<td height="42" style="height:42px;width:149px;">In-Vivo Imaging System</td>
			<td style="width:235px;">PerkinElmer</td>
			<td style="width:96px;">IVIS Spectrum</td>
			<td style="width:172px;">used for bioluminescence detecion</td>
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		<tr height="42">
			<td height="42" style="height:43px;width:149px;">&#160;isoflurane</td>
			<td style="width:235px;">Hebei yipin chemical reagents&#160; company</td>
			<td style="width:96px;">O21400</td>
			<td style="width:172px;">anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">1 ml syringe</td>
			<td style="width:235px;">Becton,Dickinson and&#160; Company</td>
			<td style="width:96px;">A257</td>
			<td style="width:172px;">cell injection</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:49px;width:149px;">digital caliper</td>
			<td style="width:235px;">Shang Hai Shen Han Measuring Tools Co., Ltd.</td>
			<td style="width:96px;">S-H</td>
			<td style="width:172px;">volume measurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">tramadol</td>
			<td style="width:235px;">Mundipharma company</td>
			<td style="width:96px;">-</td>
			<td style="width:172px;">pain killer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">D-luciferin</td>
			<td style="width:235px;">Gold Biotechnology Inc.</td>
			<td style="width:96px;">LUCK-1G</td>
			<td style="width:172px;">used for bioluminescence detecion</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:52px;width:149px;">The primary antibody against cluster of differentiation (CD) 31</td>
			<td style="width:235px;">Abcam</td>
			<td style="width:96px;">&#160;ab28364</td>
			<td style="width:172px;">used for MVD detection</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:149px;">hematoxylin and eosin staining kit</td>
			<td style="width:235px;">&#160;Beijing Zhong Shan Jinqiao Biotechnology Co., Ltd.</td>
			<td style="width:96px;">ZLI-9615</td>
			<td style="width:172px;">histology&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">hair removal cream</td>
			<td style="width:235px;">Veet</td>
			<td style="width:96px;">-</td>
			<td style="width:172px;">hair removal cream</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:149px;">Carbomer Eye Gel</td>
			<td style="width:235px;">Dr.GerhardMannChem-Pharm.FabrikGmbH</td>
			<td style="width:96px;">-</td>
			<td style="width:172px;">ophthalmic ointment</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;">sewing needle</td>
			<td style="width:235px;">Shanghai Pudong Jinhuan Medical Products Co., Ltd.&#160;</td>
			<td>17U0302J</td>
			<td style="width:172px;">suture</td>
		</tr>
	</tbody>
</table>

orthotopic injection of breast cancer cells into the mice mammary fat pad

适当的动物模型对于更好地了解疾病至关重要。通过不同的方法 (皮下注射、异种移植、基因操作、化学试剂诱导等) 建立的动物模型具有不同的病理特征, 在调查某些方面发挥着重要作用。疾病。虽然没有一个单一的模型能够完全模仿整个人类疾病的进展, 但具有适当的基质环境的原位器官疾病模型在了解疾病和筛选潜在药物方面发挥着不可替代的作用。在这篇文章中, 我们描述了如何植入乳腺癌细胞在乳房脂肪垫在一个简单, 侵入性较低, 易于处理的方式, 并跟踪转移到遥远的器官。由于原发肿瘤生长、乳腺和病变的适当特征 , 以及其他器官转移的高发生率 , 该模型最大限度地模仿了人类乳腺癌的进展。利用该模型可以研究乳腺癌的原发肿瘤原位生长、远距离转移和肿瘤微环境。

注射成功后 , 可以观察到一个白色透明的扁平球体 , 奶嘴以为外表面圆的中心 (图 1) 。原发肿瘤生长可以通过肿瘤体积和活肿瘤细胞的生物发光来衡量 (图 2)。在切除前的实验中, 肿瘤体积和总通量都增加了。在早期, 由于没有发生继发性肿瘤或原发肿瘤的强生物发光信号阻碍了微弱信号小转移病灶的检测, 因此无法找到转移。?...

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad at JoVE.com 

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad (Video) | JoVE 

在这里, 我们提出了一个协议, 植入乳腺癌细胞在一个简单, 侵入性较低, 易于操作的方式, 和这个小鼠原位乳腺癌模型具有适当的乳房脂肪垫环境, 可以用来调查各个方面的癌症。

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

这种方法可以帮助回答癌症领域关于微环境如何支持肿瘤生长或特定药物是否可以有效抑制乳腺癌进展的关键问题。肿瘤诱导技术的主要优点是无需手术，在原位建立乳腺癌是一种侵入性较小的方法。使用这种技术，肿瘤细胞生长在乳腺脂肪垫内，在适当的组织环境中，模仿人类乳腺癌的进展比皮下注射或异种移植到小鼠细胞更密切。我们的技术员曹克欣将协助张甘林演示这个程序。在手术前一天，手动约束接收小鼠，将动物腹部侧转向毛皮，在第四个奶嘴周围剃光。用薄薄的一层脱毛霜擦拭毛皮。30 至 60 秒后，使用蒸馏水取出奶油，然后用软纸巾擦干鼠标。手术当天，将乳腺癌细胞悬浮液稀释至每毫升PBS浓度的2倍至第5个细胞，在冰上无菌微离心管中。每次注射前，加载一毫升注射器，配备45比16针，每只小鼠50微升细胞。并确认每个静种动物对手趾捏缺乏反应。将软膏涂抹在动物的眼睛上，用钳子将第一只小鼠的第四个奶嘴周围的皮肤搭上帐篷，为注射器的跟随创建一个通道。握住针头朝上，皮下进入皮肤的针头从奶嘴5至10毫米，在隧道后，直到针尖几乎到达奶嘴。使用钳子在第一个奶嘴周围对皮肤进行帐篷，为注射器的跟随创造了一个通道。针头应在钳子尖之间输入，并应该抬起，而不是压在组织中。小心地将针头向上移入乳腺脂肪垫，直到斜角在奶嘴下，并释放皮肤帐篷，让癌细胞缓慢注射。插入时，小心地将针头向上移入乳腺脂肪垫，直到斜面位于奶嘴下。你会感到阻力，以确保细胞传递到正确的位置。注射所有细胞后，稍微将针头转向一侧，然后缓慢地收回注射器。使用棉签对针伤口施加压力10至20秒，使无液体渗漏。注射的奶嘴应显示为透明、白色、扁平的球体，奶嘴位于中心。要建立肿瘤体积，请将小鼠腹部侧向上，并使用卡钳测量肿瘤的长度和宽度。为了捕捉肿瘤的生物发光，在成像前10分钟，每公斤D-露西费林注射到小鼠颈部的后面150毫克。并在适当的生物发光成像参数下捕获原发肿瘤和转移的生物发光图像。在适当的实验时间点，用剪刀从皮肤上收获肿瘤。将镇痛溶液应用于手术现场，以减轻术后疼痛。然后根据标准方案用手术缝合线关闭皮肤。让动物在加热灯下恢复，并监测，直到完全恢复。使用手术刀将原发性肿瘤切成两个相等的部分。将一半组织储存在4%的甲状甲醛中，用于随后的血氧素和 eosin 染色和免疫组织化学。将另一半冷冻在液氮中，用于西印或RNAi隔离。在适当的实验终点收获肺根据标准解剖方案，并在PBS中清洗器官。然后将肺部固定为4%的甲醛，以验证血氧素和 eosin 染色的转移。如证明，原发性肿瘤生长可以通过肿瘤体积和活肿瘤细胞生物发光来测量。在接种的早期阶段，由于没有建立继发肿瘤，或者原发性肿瘤的强生物发光信号阻碍用弱信号检测小转移性肿瘤，因此不会观察到转移。切除后，可以检测和分析远器官转移性分子的生物发光信号。通过血氧林和 eosin 染色对原发性乳腺肿瘤和肺部分的形态进行分析，发现肿瘤细胞接种动物的血管生成增加，抗CD31微维塞尔标记染色就证明了这一点。尝试此程序时，请务必在传递肿瘤细胞之前确保针尖位于正确的位置。按照这个程序，雾化器，如动物超声波，可以执行，以回答有关膀胱补充原发性肿瘤在体内的其他问题。该技术开发后，为肿瘤发生领域的研究人员和药物开发领域的研究人员探索肿瘤发生的关键因素和途径以及穆林动物模型中抗癌药物的机据铺平了道路。不要忘记，使用异氟兰或其他气体麻醉剂可能极其难以剂量，并且在执行此程序时应始终采取预防措施，如使用活性炭面膜。

Research

JoVE Journal

Cancer Research

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures (Video) | JoVE 

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer (Video) | JoVE 

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish (Video) | JoVE 

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal (Video) | JoVE 

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland (Video) | JoVE 

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model (Video) | JoVE 

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad (Video) | JoVE 

大鼠乳腺内皮细胞移植到间膜白脂肪垫

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer (Video) | JoVE 

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells (Video) | JoVE 

通过炎症性乳腺癌细胞的尾静脉注射模拟脑转移

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research (Video) | JoVE 

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...