Summary

原位膀胱癌模型的基因传递研究

Published: December 01, 2013
doi:

Summary

植入癌细胞进入器官来源可以作为一个有用的临床前模型来评估新疗法。 MB49膀胱癌细胞可在膀胱内以下膀胱灌注生长。该协议表明了鼠标膀胱肿瘤植入和腺病毒转染的目的导尿。

Abstract

膀胱癌是泌尿生殖道和新的治疗方法,可以减少复发和进展所需要的第二种最常见的癌症。肿瘤微环境中可以显著影响肿瘤的发展和治疗反应。因此,它通常是可取的生长的肿瘤细胞中生成它们的器官。本协议描述膀胱癌原位模型,其中MB49小鼠膀胱癌肿瘤细胞通过导管灌入膀胱。在这个模型中成功的肿瘤细胞植入需要保护的葡糖胺聚糖层,其可通过物理或化学的方法来完成的中断。在我们的协议膀胱用胰蛋白酶处理前细胞滴注。膀胱的导管也可用于递送治疗剂,一旦肿瘤被建立。这个协议描述,表达荧光素酶报告基因的腺病毒构建体的传递。虽然Ø乌尔协议进行了优化的短期研究,侧重于基因传递,小鼠膀胱导尿的方法具有广泛的应用。

Introduction

膀胱癌是泌尿生殖道有近75,000预计在2012年1新病例和15,000死亡的第二位最常见的癌症。高复发率需要终身随访,这使得膀胱癌治疗最昂贵的癌症之一。膀胱癌已侵及肌层可能转移到肝脏,通过淋巴系统肺或骨。晚期肿瘤导致仅5年后20-40%的生存多式联运治疗。因此,迫切需要有效的治疗策略旨在减少浅表性膀胱癌的复发和进展,以及改善治疗效果的晚期患者。

新型疗法的发展需要临床前模型下初步的体外评估,以评估疗效。肿瘤微环境中可以显著影响癌症的发展和响应能力,从而强调了需要preclinic在肿瘤的发生或人的模型可以建立在器官来源。一种方法是转基因模型中的肿瘤自发产生的或可诱导的器官特异性的方式发展。转基因膀胱癌模型的一个很好的协议,已出版2。转基因模型的缺点是肿瘤倾向于慢慢地和以比希望的少的均匀发展。此外,保持了繁殖群体的成本,必须考虑到。另一种转基因模型是肿瘤细胞原位移植,其中有很短的时间帧的肿瘤成立市售小鼠的利益。虽然一些人膀胱癌细胞系可原位生长(我们已成功地使用UM-UC-3),它​​可能是可取的,以建立肿瘤的免疫活性小鼠。两种鼠膀胱癌细胞系,其中原位生长是MBT-2和MB49 3。由于MBT-2细胞被污染的复制C型逆转录病毒4,我们选择MB49细胞进行我们的研究。要注意,MB49细胞从雄性小鼠中分离和原位植入是在雌性小鼠中进行解剖结构的原因是重要的。这有便于识别的植入细胞的Y染色体标记的好处,但性别不匹配可能是一个缺点的免疫学研究。

膀胱上皮是由葡糖胺聚糖(GAG)层,其功能是作为由微生物屏障感染内衬。此屏障也可以用肿瘤细胞和几种方法植入干扰已被开发来克服这一困难( 表1)。电灼已被广泛用作物理手段破坏GAG层5-13和协议证明电灼最近已公布在朱庇特的14。但是,如果一个电灼单元不可用,化学手段破坏对GAG层如硝酸银或聚-L-赖氨酸也可以使用15-24。肿瘤是由膀胱 ​​的短暂暴露于少量体积的硝酸银有效地建立(5-10微升,0.15-1.0 M,〜10秒),或用聚-L-赖氨酸的较长的接触(100 微升的0.1毫克/毫升为20分钟)( 表1)。在这里,我们描述了使用胰蛋白酶,以促进MB49细胞植入的方法。

在试图改善的治疗方法为膀胱癌,基因疗法已经获得了显著关注。从临床上看,膀胱癌基因治疗由于容易获得器官的能力和在本地提供有效载荷的理想目标。已被研究用于膀胱癌基因治疗的病毒载体包括一种溶瘤单纯疱疹病毒25,逆转录病毒26,金丝雀痘病毒27,牛痘病毒,腺相关病毒,和adenoviru第28号。在我们的协议的第二部分中,我们描述了用于病毒递送,几乎是相​​同的肿瘤细胞的灌注的方法。在我们的实验室中感兴趣的是新颖的方法,以基因递送,我们使用表达萤光素酶转基因的腺病毒载体通过生物发光评估的发展。然而,可用于输送各种试剂膀胱导管的方法,因此具有广泛的适用性。

Protocol

所有涉及动物的程序进行了审查和批准的机构动物护理和使用委员会在南卡罗来纳医科大学。该协议是根据美国农业部的D类止痛批准。 1。细胞移植在执行程序前两天,板1×10 6 MB49细胞转化为T-25瓶。使用添加有10%FBS(和抗生素,如果需要的话)高糖DMEM。一个烧瓶中足以为每个组最多5只小鼠将被麻醉并植入平行。 在手术当天,通过用无菌DMEM基础培养?…

Representative Results

血尿是在几乎所有的小鼠中观察到内200,000 MB49细胞植入后8天。在非肿瘤小鼠为87.5±19.2毫克(范围77-120毫克,N = 10) 如图1,膀胱重量比双打更从34.7±3.3毫克(范围31-37毫克,N = 4),在小鼠已经植入MB49细胞。在基因传递方面,我们发现,小鼠成像24小时后病毒滴入产生更强的信号超过48小时( 图2)之后。腺病毒转染的动物,它应该用于统计目的的策划组大小时,必须考虑…

Discussion

在本协议中所述的主要方法是导尿鼠标膀胱,这对细胞的灌注或用于本地传递到膀胱上皮任何代理广阔的应用前景。上文所述的特定的协议进行了优化的短期研究(〜10日)。植入细胞的精确数目是至关重要的,因为更高的细胞数,将导致更快速的肿瘤生长和可能的动物的损失是由于肿瘤负荷大。使用200,000 MB49细胞滴注可能需要高达实施安乐死的动物的25%由14天因过度肿瘤负荷。根据我们的经验?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国国立卫生研究院R21 CA143505克里斯蒂娜Voelkel – 约翰逊的支持。

Materials

Name of reagent

Company

Catalog number

Comments

6-8 week old female mice

Jackson Laboratories

Strain Name: C57BL/6J

Stock Number: 000664

Trypsin*

MediaTech

MT25-053-CI

Obtained through Fisher

DMEM*

MediaTech

MT10-017-CV

Obtained through Fisher

FBS

Hyclone

SH30071.03

Heat-inactivated

T25 flasks*

Corning Costar

Corning No.:3056

Fisher: 07-200-63

Obtained through Fisher

MB49 cells

N/A

N/A

Obtained from Dr. Boehle (see reference11)

Puralube Vet Ointment*

Pharmaderm

Henry Schein Company

No.:036090-6050059

Fisher: NC9676869

Obtained through Fisher

Depilatory cream: Veet

local pharmacy

Lubricant:

K-Y Jelly

local pharmacy

Catheters*

Exel International

Exel International

No.:26751;

Fisher: 14-841-21

Obtained through Fisher

Isoflurane

Terrell

NDC 66794-011-25

Obtained though hospital pharmacy

1 ml slip tip TB syringes

Becton Dickinson

BD309659

Fisher:14-823-434

D-Luciferin

Gold Biotechnologies

L-123-1

Ad-CMV-Luc

VectorBiolabs

1000; Request large scale amplification and CsCl purification for in vivo use

Infectious agent that requires BSL2 containment

Steady-Glo Luciferase Assay System

Promega

E2510 (10 ml), E2520 (100 ml), or E2550 (10 x 100 ml)

*available through multiple vendors

EQUIPMENT

Material name

Company

Catalog number

Comments

Anesthesia system

E-Z Systems, Euthanex Corporation

Anesthesia system: EZ7000

5-port mouse rebreathing device: EZ109

Obtained through Fisher

Xenogen IVIS 200

Caliper Life Sciences

http://www.caliperls.com/products/preclinical-imaging/ivis-imaging-system-200-series.htm

FLUOstar Optima

BMG Labtech

http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2

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Cite This Article
Kasman, L., Voelkel-Johnson, C. An Orthotopic Bladder Cancer Model for Gene Delivery Studies. J. Vis. Exp. (82), e50181, doi:10.3791/50181 (2013).

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