Name: intra-cardiac injection of human prostate cancer cells to create a bone metastasis xenograft mouse model
Uploaded: 2022-11-04T14:30:00
Description: Watch this Scientific Journal Video about Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model at JoVE.com

这项工作得到了国家重点研发计划（2018YFC1704300和2020YFE0201600）、国家自然科学基金（81973877和82174408）、上海中医药大学预算内的研究项目（2021LK047）和上海市医院中药制剂产业转化协同创新中心的资助。

6至8周龄雄性BALB / c无胸腺小鼠（n = 10）在12小时光照/黑暗循环条件下，在无特异性（SPF）动物室中单独通风的小鼠笼子（5只小鼠/笼）中，自由获得SPF饲料和无菌水。小鼠在实验前适应性喂养一周。所有动物实验均经上海中医药大学动物福利委员会批准。
1. 细胞制备
<ol><li>在前列腺癌细胞注射当天，用预冷无菌PBS（pH 7.4）洗涤在10cm细胞培养皿中培养的80%-90%汇?...

<ol>
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心内注射人前列腺癌细胞产生骨转移是探索前列腺癌骨转移的功能和机制以及评价治疗效果的理想小鼠模型。研究表明，骨损伤最有可能发生在胫骨近端和股骨远端17，这可能是由于它们的高血管化和代谢活性。
由于骨转移是乳腺癌患者中经常观察到的转移性病变，因此通过心内注射乳腺癌细胞产生的骨转移模型也常用于乳腺癌研究18?...

前列腺癌是112个国家中男性最常见的癌症，在人类发展指数较高的国家中排名第二1，2。前列腺癌患者的死亡大多是由转移引起的，约65%-75%的病例会发生骨转移3，4。因此，迫切需要预防和治疗前列腺癌骨转移，以改善前列腺癌患者的临床结果。骨转移动物模型是探索前列腺癌骨转移各阶段涉及的多阶段过程和分子机制，从而确定治疗靶点和开发新疗法不可或缺的工具5。
生成前列腺癌骨转移实验动物模型的最常见方法包括原位、骨干内（如胫骨内）和心内注射前列腺癌细胞。原位注射的骨转移模型是通过将前列腺癌细胞直接注射到小鼠的前列腺中而生成的6，7。该实验动物模型与前列腺癌骨转移具有非常相似的临床特征。然而，转移主要发生在腋窝淋巴结和肺，而不是骨8，9。前列腺癌的胫内注射模型直接将前列腺癌细胞注射到胫骨中，骨（胫骨）肿瘤形成率高10，11;但是，骨皮层和骨髓腔很容易受损。此外，胫骨注射方法不能刺激前列腺癌骨转移的病理过程，其中癌细胞通过循环定植骨骼。为了研究癌细胞骨转移率较高的循环、血管外渗和远处转移，已经开发出一种通过将前列腺癌细胞直接注射到小鼠左心室的心内注射技术8，12，13。这使其成为骨转移研究的有价值的动物模型8。心内注射法显示骨转移率约为75%9，14，远高于原位注射法。因此，心内注射是生成前列腺癌骨转移动物模型的理想方法。
这项工作旨在描述建立前列腺癌骨转移小鼠模型的过程，让读者可视化模型的建立。目前的工作提供了详细的过程，预防措施和说明性图片，以通过在无胸腺小鼠中心内注射人前列腺癌细胞 来 生成骨转移异种移植模型。该方法为进一步探索前列腺癌骨转移的分子机制和 体内 治疗效果提供了有效的工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringes and needles</td>
			<td>Shandong Weigao Group Medical Polymer Co., Ltd</td>
			<td>20200411</td>
			<td>The cells were injected into the ventricles of mice</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>R500IP</td>
			<td>Equipment for anesthetizing mice</td>
		</tr>
		<tr>
			<td>Automatic cell counter</td>
			<td>Shanghai Simo Biological Technology Co., Ltd</td>
			<td>IC1000</td>
			<td>&#160;For counting cells</td>
		</tr>
		<tr>
			<td>BALB/c athymic mice</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd.</td>
			<td>Male</td>
			<td>6-8 week old, male mice</td>
		</tr>
		<tr>
			<td>Bioluminescence imaging system</td>
			<td>Shanghai Baitai Technology Co., Ltd</td>
			<td>Vieworks</td>
			<td>For tracking the tumor growth and pulmonary metastasis if the injected cells are labeled by luciferase</td>
		</tr>
		<tr>
			<td>Centrifuge tube (15 mL, 50 mL)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>&#160;430790, Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Wuhan Xavier Biotechnology Co., Ltd</td>
			<td>G1105</td>
			<td>&#160;For decalcification of bone tissure</td>
		</tr>
		<tr>
			<td>F-12 medium</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>21700075, GIBCO</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>BL539A</td>
			<td>For fixing the specimen of each mouse</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>VETEASY</td>
			<td>For anesthesia&#160;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>11668027, Thermo fisher</td>
			<td>Plasmid transfection reagent</td>
		</tr>
		<tr>
			<td>PC-3 cell line</td>
			<td>Cell Bank of Chinese Academy of Sciences</td>
			<td>TCHu 158</td>
			<td>Prostate cancer cell line</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Beyotime Biotechnology</td>
			<td>ST447</td>
			<td>Wash the human osteosarcoma cells</td>
		</tr>
		<tr>
			<td>Trypsin (0.25%)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>25200056, Gibco</td>
			<td>For detaching the cells</td>
		</tr>
		<tr>
			<td>Vector (pLV-luciferase)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>VL3613</td>
			<td>Plasmid for transfection</td>
		</tr>
		<tr>
			<td>X-ray imaging system</td>
			<td>Brook (Beijing) Technology Co., Ltd</td>
			<td>FX PRO</td>
			<td>For obtaining x-ray images to detect tumor growth</td>
		</tr>
		<tr>
			<td>&#956;CT80</td>
			<td>Shenzhen Fraun Technology Service Co., Ltd</td>
			<td>Scanco Medical AG,Switzerland</td>
			<td>For detection of bone destruction. The mico-CT is equipped with 3DCalc, cone reconstruction,&#160; and &#956;CT Ray V3.4A model visualization software.</td>
		</tr>
	</tbody>
</table>

intra-cardiac injection of human prostate cancer cells to create a bone metastasis xenograft mouse model

作为最常见的男性恶性肿瘤，前列腺癌（PC）的死亡率排名第二，主要是由于65%-75%的骨转移率。因此，了解前列腺癌骨转移的过程和相关机制对于开发新的治疗方法至关重要。为此，骨转移的动物模型是必不可少的工具。在这里，我们报告了通过心内注射前列腺癌细胞 来 生成骨转移小鼠模型的详细程序。生物发光成像系统可以确定前列腺癌细胞是否已准确注射到心脏中并监测癌细胞转移，因为它在监测转移性病变发展方面具有很大的优势。该模型复制播散癌细胞的自然发育，在骨骼中形成微转移，并模仿前列腺癌骨转移的病理过程。为进一步探索本病的分子机制和 体内 治疗效果提供了有效的工具。

生物发光成像在监测心内注射模型的转移性病变发展方面具有巨大的优势。癌细胞注射后不久（24小时内），使用生物发光成像来可视化进入一般循环的癌细胞（图3A）。当癌细胞被正确注射到动脉循环中时，全身都会看到明显的生物发光信号。来自仅在注射部位（心脏）显示生物发光信号的小鼠数据应从最终数据收集中排除。细胞注射后2周观察到后肢的转移性病变（<strong ...

Watch this Scientific Journal Video about Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model at JoVE.com

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

在这里，我们提出了一种用于心内注射人前列腺癌细胞的方案，以生成具有骨转移病变的小鼠模型。

心内注射人前列腺癌细胞以创建骨转移异种移植小鼠模型

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

该方法可以帮助产生一个理想的模型，该模型密切模拟体内前列腺癌细胞骨转移的自然进展。该技术具有骨转移的优点，并且代表了前列腺癌细胞在体内的自然骨转移过程。该技术有利于探索前列腺癌骨转移的分子机制和体内治疗效果的研究。在注射当天，取在10厘米细胞培养皿中培养的80至90%融合荧光素酶标记的PC3细胞，并通过用冷无菌PBS洗涤两次开始准备注射细胞。用1.5毫升0.25%胰蛋白酶消化洗涤的细胞三分钟，然后通过加入6毫升含有10%血清的F12培养基来淬灭胰蛋白酶，并将细胞收集到15毫升离心管中。将20微升收集的细胞悬液转移到细胞计数板中，并使用自动细胞计数器计算细胞浓度。接下来，在室温下以800G离心细胞五分钟.使用移液管丢弃上清液并将细胞沉淀重悬于F12培养基中以达到所需的最终细胞密度。现在将细胞带到手术室并在两小时内进行细胞注射。将细胞保持在冰上直到注射。要开始手术，将麻醉的小鼠置于仰卧位，并通过垂直拉伸它们远离中线来固定两个上肢。使用手术胶带，将鼠标从腹部向下固定，不要用力按压并移位内脏。使用70%乙醇棉签对胸部皮肤进行消毒。接下来，触诊前胸壁的中间，将小鼠剑突定位在胸骨中部最低端的凹陷处。标记剑突的最下点。然后从前胸壁中间向上触诊，将小鼠颈静脉切口定位在手掌上缘的中央凹陷处，并标记颈静脉切迹的最下点。现在找到并标记前两个标记之间的中点，稍微向右，正好在第三个肋间隙的心脏上方。这是注入点。在将200微升悬浮液装入装有26号针头的1毫升注射器中之前，彻底混合细胞悬液。在柱塞和悬浮液之间的注射器中留出一些空气，以允许注射过程中脉动血液进入。要进行注射，请将针头垂直插入注射部位。如果针尖正确插入左心室，针尖中将可见鲜红色的脉动血液。如果血脉动不可见或血液凝固，请重试注射。正确插入针头后，用稳定的手在 40 到 60 秒内非常缓慢地注射 100 微升细胞悬液。注射完成后，缩回针头，同时用干棉签对注射部位施加压力15秒，以确保止血。将鼠标放回干净的笼子并监测动物，直到它从麻醉中完全恢复。将荧光素酶标记的PC3细胞注射到小鼠中24小时后进行的生物发光成像显示来自动物全身的信号表明体循环中存在癌细胞。注射两周后，转移性病变出现在小鼠的后肢。随着时间的推移，转移性病变变大，并开始出现在其他部位。使用X射线成像进一步检测前列腺癌细胞诱导的骨骼转移性病变，该成像显示动物胫骨近端的骨破坏。显微CT扫描也证实了这一点。通过血红素和伊红染色在石蜡包埋组织中进一步证实转移性病变。准确的注射部位对于保证该模型的成功率非常重要。这可以通过实践来实现。

Research

JoVE Journal

Cancer Research

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

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

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 ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

颈动脉肿瘤细胞注射，产生脑转移瘤小鼠模型

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

同源原位骨肉瘤Sprague Dawley大鼠模型，截肢控制转移率

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

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

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 ...