Name: a murine orthotopic bladder tumor model and tumor detection system
Uploaded: 2017-01-12T15:00:00
Description: Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

坚持上新加坡国立大学动物使用和处理（协议号084/12）的机构动物护理和使用委员会（IACUC）的指导方针所有动物的工作。 1.成长MB49-PSA细胞在体外和测量PSA分泌<ol><li>保持小鼠膀胱癌细胞MB49 - PSA 15在完整的Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2毫摩尔/升L-谷氨酰胺，和0.05毫克/毫升青霉素-链霉素在培养箱在37 ℃和5％的CO 2。...

<ol>
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在该协议中最关键的步骤是：1）成功地保持了细胞系的致瘤性; 2）确保小鼠肿瘤细胞植入前测PSA的分泌; 3）产生用于植入的单细胞悬浮液，从而减少肿瘤大小的变化;和4）灌输细胞以缓慢的速度，以防止膀胱输尿管回流，导致在肾肿瘤细胞植入。 体外延长传代后，MB49 / MB49 - PSA细胞可失去其致瘤性，这表现在它们不能在小鼠中产生肿瘤。这可能是一个混杂因素，以是?...

该方法的目标是产生鼠正位膀胱肿瘤，并尽可能准确地监测植入的肿瘤，所以没有肿瘤植入的小鼠都没有想到在终点分析已经固化。总体上，所示的方法将减少大量小鼠实验分析的需要，并确保在确定治疗结果更高的精度。 对于癌症的原位模型的发展是重要的，因为植入肿瘤细胞皮下不概括的临床疾病的环境或启用的治疗策略的开发。膀胱的结构允许膀胱癌治疗的滴注直接进入以最小的全身效应膀胱。因此，概括这种环境的动物模型，例如原位模型，是很重要的，以评估新疗法。从任何实验装置得出的结论是dependen在该模型的局限性吨。 几种技术已经开发了用于在小鼠中生产原位膀胱肿瘤。这些依赖于损伤膀胱的糖胺聚糖层，使肿瘤细胞被植入。所使用的技术包括电烙，这导致在膀胱壁损伤的单点，在膀胱1,2-导致肿瘤发展在一个地点。然而，使用电灼肿瘤植入的成功率是取决于运营商从10变 - 90％，并且它包括危险，即膀胱壁将被删截，从而导致肿瘤在腹腔显影。化学烧灼是用硝酸银，这损害了膀胱壁3进行。同样，酸已被用来损伤膀胱壁4。胰蛋白酶也被用来损伤膀胱以及5。这些方法可能会导致在一个以上的肿瘤在膀胱的发展。此外，存在严重损坏的膀胱的危险，如果化学品留在与膀胱壁接触时间过长。通过Ninalga 等人开发的方法。使用带正电荷的聚-L-赖氨酸（PLL）6分子涂覆膀胱壁;这使得带负电荷的肿瘤细胞粘到膀胱的糖胺聚糖层。这种方法通常导致在一个以上的肿瘤在膀胱显影，但肿瘤植入是在80 - 100％的4,7。从技术上讲，它也是以执行的最简单的过程。以保证该开发肿瘤即使在大小相当，它是重要的肿瘤细胞中不植入前大团块分组。 为了评估治疗功效，最好是具有相当类似大小的肿瘤对小鼠进行这项研究。因此，可以在植入后不久量化肿瘤大小良好的检测系统是重要的。几种策略有蜜蜂用于n评估肿瘤。这些包括磁共振成像（MRI）8-10，荧光11，生物发光12,13，超声14，和酶联免疫吸附试验（ELISA）15,16。而MRI和超声不需要的肿瘤细胞中的修改，有必要对敏感设备和造影剂用于MRI。的荧光 - ，luminescence-，和基于ELISA测定所需要的肿瘤细胞的修饰来表达，可以通过这些方法来检测标志物蛋白。为发光，需要用于检测荧光素酶活性的基板;因此，有一个附加的步骤，增加成本。这两个发光和荧光需要专门的设备。为了产生荧光，绿荧光蛋白（GFP）的环化，它是由分子氧催化的，是必需的。因此，GFP表达可能会因访问氧肿瘤块内的变量，使之成为一个相当可靠的指标<sup&gt; 17。鼠膀胱癌细胞系MB49的修饰分泌人类前列腺特异性抗原（PSA）的15,16作为替代标志物是另一种策略。这些标志物还提供确认肿瘤存在在实验结束时，使它们的替代免疫组织化学的替代手段。这项研究报告原位肿瘤植入的PLL方法，并提出肿瘤检测系统，即ELISA，荧光和超声成像的比较。

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:404px;">MB49-PSA cells</td>
			<td style="width:188px;">N/A</td>
			<td style="width:188px;">N/A</td>
			<td style="width:113px;">ref (Wu QH, 2004)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640 media</td>
			<td>HyClone</td>
			<td>SH30027.01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media</td>
			<td>Biowest</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American, Premium</td>
			<td>Biowest</td>
			<td>S181B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30088.03&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine</td>
			<td>Biowest</td>
			<td>X0550</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin-Streptomycin</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hygromycin B</td>
			<td>Invitrogen</td>
			<td>10687-010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">free PSA (Human) ELISA kit</td>
			<td>Abnova</td>
			<td>KA0209</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TRIzol Reagent for RNA extraction&#160;</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>4374967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Mouse Actb</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Mm00607939_s1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Human KLK3</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Hs00426859_g1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C57BL/6J female mice</td>
			<td>In Vivos</td>
			<td></td>
			<td>4-6 wk old</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reversal drug (1mg/kg Atipamezole)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ear punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>72893-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hartmann&#39;s solution or Compound sodium lactate</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat pack - HotHands handwarmers</td>
			<td>Heatmax Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Introcan Certo IV catheter</td>
			<td>B Braun</td>
			<td>4251300</td>
			<td>24G x 3/4&#8243;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aquagel Lubricating jelly</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;Poly-L-lysine solution, 0.01%,</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Quantichrom Creatinine Assay Kit</td>
			<td>BioAssay Systems</td>
			<td>DICT-50&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescent dye - VivoTrack 680</td>
			<td>Perkin Elmer</td>
			<td>NEV12000&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNAlater-ICE Frozen Tissue Transition Solution</td>
			<td>Ambion</td>
			<td>4427575</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Realtime PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Software v2.3</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Metabolic Cage</td>
			<td>Tecniplast&#160;</td>
			<td>Vertical type rack for 12 cages</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSCanto I system&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSDiva software v7</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IVIS SpectrumCT in vivo imaging system&#160;</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Living Image Software v3.1</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vevo 2100 imaging system&#160;</td>
			<td>VisualSonics&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine orthotopic bladder tumor model and tumor detection system

这个协议描述膀胱肿瘤的生成中使用鼠膀胱癌细胞系MB49，其已被修饰以分泌人类前列腺特异性抗原（PSA）的雌性C57BL / 6J小鼠，对于肿瘤植入的确认的步骤。简言之，老鼠是使用注射药物麻醉，由背侧位置埋下了伏笔。尿从膀胱腾空和聚-L-赖氨酸（PLL）的50微升缓慢以10微升/ 20秒的速率用24克IV导管灌输。它是由塞住导管留在膀胱20分钟。导管被移除和PLL是通过在膀胱温和的压力腾空。这之后是鼠膀胱癌细胞系以10微升/ 20秒的速度（1×10 5个细胞/50μl）滴注。导管被塞住，以防止过早疏散。 1小时后，将小鼠恢复与逆转药物，和膀胱腾空。缓慢滴注速度是很重要的，因为它减少膀胱输尿管回流，这可能会导致肿瘤在上尿路和在肾脏发生。细胞系应该很好再悬浮，以减少细胞的聚集，因为这可能在植入后导致不均匀肿瘤大小。 这种技术诱导效率高的肿瘤。肿瘤生长是由泌尿系PSA分泌监控。 PSA标志的监测比超声波或荧光成像用于检测肿瘤的膀胱中存在的更可靠。如果不及时治疗4周 - 小鼠肿瘤约3一般达到最大大小负面影响健康。通过监测肿瘤的生长，有可能区分那些未成功地与肿瘤植入被治愈的小鼠。只有终点分析，后者可被误认为已通过疗法治愈。

从MB49细胞的PSA分泌被发现与生长培养基而变化。 MB49 - PSA是在DMEM培养基中生长，因为这导致增加的PSA的分泌（ 图1A）。为了确定所述PSA ELISA和实时PCR的灵敏度，MB49 - PSA分泌细胞的不同数目的用MB49亲代细胞混合。 PSA ELISA检测到至少1×10 5个的PSA分泌细胞/ 1×10 6个细胞（ 图1B），而实时PCR分析可以检测100的PSA分泌细胞/ 1×10 6个</...

Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

这个协议描述在雌性C57BL / 6J小鼠和肿瘤生长的监测小鼠原位膀胱肿瘤的生成。

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Research

JoVE Journal

Cancer Research

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

科研

癌症研究

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. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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 ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

利用小鼠脑肿瘤球的原发染色质沉淀

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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 ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...