这项工作是由意大利ASSOCIAZIONE每拉RICERCA SUL巨蟹座，大学意大利和研究部，首席项目和PROGETTI迪RICERCA雅典耀迪，给予N°CPDA137871，基金会Cariplo支持，给予N°2011-0485到MDB，并通过Finanziamento乔瓦尼Studiosi，帕多瓦大学，盖亚Codolo。

所有的程序，动物处理已批准由意大利卫生部（DM二千○一十一分之二百○四-B）。 1.动物<ol><li>生长C57BL6 / J雌性小鼠8周龄在单独通风笼了显微过滤器。 注：女性是首选，因为其外部泌尿生殖设备的解剖形态呈现比男性导尿更容易。 </li></ol> 2.细胞培养<ol><li>保持小鼠尿路上皮癌的细胞系MB49在RPMI 1640培养基中在潮湿的5％CO 2中在补充有10％热灭活的胎牛血清和50μg/ ml庆大霉素，37℃。 </li><li>生长在T-75烧瓶至80％汇合MB49细胞。 Trypsinize重悬在0.5×10 5 -植入小鼠前0.5×10 6细胞每150微升PBS中。 </li></ol> 3.膀胱肿瘤植入<ol><li>麻醉使用的麻醉剂zoletil和xylor的混合物（33毫克/公斤和20毫克/公斤，分别）小鼠，腹膜内注射。 </li><li>将动物仰卧位和固定后腿利用胶带垫。爪子已被充分分开，以使尿道口以及可见和对导管的插入很方便。 </li><li>注：对于导尿，一个24克小儿静脉导管适合8至10周大的老鼠。导管的选择是，为了避免液体的尿道壁和导管之间的泄漏很重要。较大的孔导管必须用于更大或更老的小鼠。 </li><li>用一个小镊子，捏尿道外部分的一个边缘和扭曲它轻轻地向“前端”的老鼠。这个动作暴露尿道口。 </li><li>取出导管的内部针和插入后者在尿道以45°的角度，略微取向对老鼠的“尾部”。不要强迫导管的入口;在遇阻力，只是扭动导管轻轻地，直到它滑倒尿道内。当导管为约0.5-0.8厘米是在尿道，小心地将平行导管给小鼠的尾部并完全插入。 </li><li>使用1ml注射器，针，其中，必须在导管插入时，通过注入200-300微升无菌PBS洗涤从残余尿膀胱和吸从膀胱所有的液体。 </li><li>注意：当注射的PBS，膀胱将填满和肿胀将小鼠的后腿之间可见;这证实了导尿是正确的。大多数导管有必须计算进样量时，被认为是一个死体积。 </li><li>使用无菌1ml注射器，注入200微升的0.1N HCl中。填补了膀胱，以燃烧的整个墙面。经过10秒，除去所有的酸，并立即neutrali泽通过注入200微升0.1N NaOH中，用无菌注射器。在注射的最高数目的细胞（0.5×10 6）的，则不需要酸化步骤。 </li><li>抽吸所有的残余液体，并立即冲洗腔用300μl无菌PBS中。 </li><li>排空膀胱吸，并注入150微升PBS含MB49细胞（范围0.5×10 5 - 0.5×10 6）进入膀胱。离开组装到导管，以避免细胞的泄漏的注射器。最好是通过将其固定在利用胶带垫以固定注射器。 </li><li> 1小时后，轻轻地从尿道中提取的导管，然后将小鼠在笼下的白炽灯，直到他们醒着：这通常发生1至2小时的治疗结束后。 </li></ol> HP-NAP 4.膀胱交货<ol><li>注：细胞滴注后至少3天，有可能开始治疗。 </li><li>导尿与步骤3.1至3.4中所述。在这个阶段，没有必要用HCl烧膀胱壁。 </li><li>使用1ml注射器，通过注入200-300微升无菌PBS洗涤从残余尿膀胱和吸从膀胱所有的液体。 </li><li>注入50％的150微升PBS HP-NAP微克进入膀胱。离开装在导管的注射器，以避免该蛋白质溶液的泄漏。将它固定在用透明胶带固定垫注射器。 </li><li> 1小时后，轻轻地提取从尿道导管，然后将小鼠在笼下的白炽灯，直到他们清醒。 </li></ol> 5.肿瘤外植体及组织学分析<ol><li>在治疗结束时，牺牲的动物，将它们放置在仰卧位置并固定后腿用透明胶带垫。 </li><li>使用外科手术刀，通过皮肤和使一个纵切口约1.52厘米长的腹膜，从正上方外生殖器的“前端”的老鼠。为了尽量减少破坏膀胱的风险，特别注意的切削深度。 </li><li>膀胱将被定位在切口的中间偏左和肿瘤会是可见的，为暗粉红色/紫色的质量。在其切除，使整个膀胱组织学笼和石蜡包埋前在10％缓冲福尔马林固定。 </li><li>对于组织学和免疫组织化学分析，准备使用标准切片膀胱（46微米厚）的连续切片。 </li><li>染色用苏木精/曙红的部分，并继续进行与肿瘤大小（D xd的2），并确定每使用计算机辅助图像分析仪截面积坏死的百分比的计算。 </li><li>对于血管计数，通过进行染色的部分为血管内皮标记CD31 / PECAM，使用标准的免疫过氧化物酶技术。扫描的部分的吨低功率（4×），以确定最高血管密度的区域。在这个区域，算上五个独立的随机领域的个人微血管在高功率（20×）。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

多数在癌症治疗的进展的需要测试在动物模型开始临床试验之前。研究肿瘤生物学体内 ，以动物模型的优势的可能性，表示为研究人员研究癌症发病机制，使不同的治疗方法的评估的一个重要工具。由于大量的细胞系可用于建立肿瘤模型和原位模型保持黄金标准14-15，一方面是因为它们模拟天然存在的肿瘤16的环境。 此外，设置的癌原位模型在同系小?...

膀胱癌是泌尿生殖道的最常见的癌症之一，每年有近75000例新病例，在美国1。高复发率需要终身随访，这使得膀胱癌治疗的最昂贵的癌症之一。金标准治疗的高档NMIBC是经尿道切除术，随后膀胱内免疫卡介苗。虽然卡介苗的抗肿瘤活性的确切机制仍有待完全阐明，人们公认的富含T辅助（TH）1和细胞毒性T（Tc）的1细胞的细胞介导的​​免疫应答的激活是关键治疗2的成功。 尽管卡介苗仍然NMIBC选择的治疗的事实，高比例的患者不给治疗的反应;此外，它可能会导致一些副作用：约70％的治疗的肿瘤的一段时间后，复发和〜15％的进展到的肌肉侵入形式疾病。 BCG与治疗有关的其他副作用包括排尿困难，膀胱炎，肾感染3-6。 新的治疗策略的发展必须考虑使用临床前模型中，下列初始体外评估;这是肿瘤微环境的可它们的发展和响应显著影响到治疗尤为重要。 在过去十年中，我们已经解决的HP-NAP，由幽门螺杆菌产生的蛋白质的免疫调节活性的多个方面，初步确定为能够促进多形核细胞（中性粒细胞）7的内皮粘附的。在结构上，HP-NAP属于下饥饿条件（DPS）家族8的DNA的保护蛋白质，由布置在一个十二聚体壳12相同的亚基。 我们已经表明，HP-NAP是Toll样受体（TLR）2激动剂，与负责驱动T的分化强烈免疫调节活性淋巴细胞朝向Th1细胞表型，无论是在体外和体内 9-10。借助此活动的，HP-NAP是能够重定向Th2免疫应答成过敏性哮喘11的鼠模型的更有利的Th1应答。 为了评价HP-NAP的Th1细胞依赖性抗肿瘤潜力，我们采取了膀胱癌的小鼠模型开发了数年前由奥唐奈和同事12用于评估BCG给药的影响的优点。 有了这个协议，我们证明了HP-NAP具有抗膀胱癌具有很强的抗肿瘤潜力，HP-NAP管理的有效性平行Th1和Tc1​​的淋巴细胞产生干扰素的显著积累，肿瘤及区域淋巴结内，（ IFN）-γ13 </SUP&gt;。肿瘤从HP-NAP处理的小鼠中分离表现出更多的坏死和比未处理对口血管较少。 本报告提供一个分步协议，详细介绍了动物的制备方法，其导尿，所需细胞附着到膀胱粘膜的化学燃烧，并在注射肿瘤细胞。我们还描述的HP-NAP可被视为对膀胱癌的治疗开发的任何治疗剂的原型的局部给药。通过比较肿瘤从控制和HP-NAP处理的动物中分离得到的证据不仅强调一个事实，即HP-NAP可能是一个很好的候选为膀胱癌免疫疗法，而且实验设置的常规效力。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>C57BL/6J female mice</td>
			<td>Harlan Italy (Udine, Italy)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MB49 Cells</td>
			<td></td>
			<td></td>
			<td>Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma-Aldrich (St. Louis, Missouri, USA)</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td>10X, to be diluted in apyrogenic water</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Becton Dickinson (Franklin Lakes, New Jersey, USA)</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>Becton Dickinson</td>
			<td>301358</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies (Waltham, Massachusetts, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Life Technologies</td>
			<td>15710-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Xilor</td>
			<td>Bio 98 s.r.l. (Milano, Italy)</td>
			<td></td>
			<td>2% Xylazin</td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>Virbac (Carros, France)</td>
			<td>359713301992</td>
			<td>5% Zolazepam + 5% Tiletamine</td>
		</tr>
		<tr>
			<td>24G Catheter</td>
			<td>Terumo (Rome, Italy)</td>
			<td>SR+DM2419PX</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carlo Erba Reagents (Milano, Italy)</td>
			<td>403871</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>JT Baker (Center Valley, Pennsylvania, USA)</td>
			<td>10095011</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Albion Surgical Limited (Sheffield, England)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Microsystem ( Wetzlar, Germany)</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR International (Radnor, Pennsylvania, USA)</td>
			<td>631-0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analyzer</td>
			<td>Zeiss (Jena, Germany)</td>
			<td>Cyres System</td>
			<td></td>
		</tr>
	</tbody>
</table>
Materials C57BL/6J female mice Harlan Italy (Udine, Italy) MB49 Cells Obtained from Prof. O&#39;Donnel, University of Iowa Carver College of Medicine, Iowa, USA RPMI Sigma-Aldrich (St. Louis, Missouri, USA) R8758 FBS Sigma-Aldrich F7524 PBS Sigma-Aldrich D1408 10X, to be diluted in apyrogenic water Flask Becton Dickinson (Franklin Lakes, New Jersey, USA) 353135 Syringe 1ml Becton Dickinson 301358 Trypsin Life Technologies (Waltham, Massachusetts, USA) Gentamycin Life Technologies 15710-049 Xilor Bio 98 s.r.l. (Milano, Italy) 2% Xylazin Zoletil Virbac (Carros, France) 359713301992 5% Zolazepam + 5% Tiletamine 24G Catheter Terumo (Rome, Italy) SR+DM2419PX HCl Carlo Erba Reagents (Milano, Italy) 403871 Liquid NaOH JT Baker (Center Valley, Pennsylvania, USA) 10095011 Powder Equipment Surgical Scalpel Albion Surgical Limited (Sheffield, England) Microtome Leica Microsystem ( Wetzlar, Germany) RM2235 Microscope Slides VWR International (Radnor, Pennsylvania, USA) 631-0108 Image Analyzer Zeiss (Jena, Germany) Cyres System

evaluation of the efficacy of the h. pylori protein hp-nap as a therapeutic tool for treatment of bladder cancer in an orthotopic murine model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

图1示出了导管插入术;鼠标插入导管和滴注0.5×10 6 MB49细胞。治疗与HP-NAP开始注射肿瘤细胞后3天，以使它们能够连接至膀胱壁和增殖。所有属于控制组动物发展肿瘤;他们中的一些13天期限结束之前可能死于尿道的阻塞。 在第一次注射与HP-NAP后，对动物进行治疗，每三天，总共四次注射。在实验结束时，在13天，将动物处死，取出气囊收集用于组织学和免?...

Watch this Scientific Journal Video about Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model at JoVE.com

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

7.2K 次观看.  University of Padua. Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.