作者希望你要感谢金耶茨，艾琳富兰克林，和丽贝卡Tjepkema（动物护理和兽医服务，渥太华大学）与动物手术援助。李华泰是由一个全宗德RECHERCHE桑特魁北克奖学金支持。领导者的机会格兰特丽贝卡奥尔 - 这项工作是由加拿大癌症协会研究所创新资助，研究与发展早期研究者奖安大略省教育部和加拿大创新基金会运作的资金支持。

1。保持4T1肿瘤细胞在体外 <ol><li>培养未修改的4T1肿瘤细胞的完全DMEM培养液（DMEM，10％FBS，1×青霉素/链霉素）的10厘米的组织培养板中。孵育在37℃，5％CO 2的细胞培养箱。 </li><li>文化分裂2-3次/周。为保持最佳活力细胞不应该超过80％汇合。不要使用细胞建立体内的原发性肿瘤，如果文化已经传代体外 &gt; 1个月，因为这降低恶性肿瘤转移潜能比早期传代细胞。 </li></ol> 2。收获4T1肿瘤细胞注射<ol><li>从用巴氏吸管的组织培养板中吸出培养基。 </li><li>加入含2ml EDTA10毫升1X无菌PBS。让的PBS溶液，在37℃，5％CO 2的组织培养箱中5-7分钟孵育的板。 </li><li>从板河收获PBS溶液insing板2-3X使用的解决方案，转移到15毫升锥形管。 </li><li>细胞离心5分钟，在500×g离心，在4℃下，在台式离心机。 </li><li>吸去上清，重悬在无血清的DMEM将细胞沉淀。 </li><li>用细胞计数测定细胞浓度。 </li><li>稀释的细胞用无血清培养基中以1×10 6 cells/500微升（1×10 5 cells/50微升（用于注射和定位细胞于冰上。 </li></ol> 3。注射小鼠4T1肿瘤细胞完成所有的动物研究是根据在渥太华建议大学动物护理兽医服务用于接收原位注射或植入肿瘤的所有动物的组织指导方针。 <ol><li>皮下注射治疗小鼠丁丙诺啡（0.05毫克/千克）手术疼痛管理前1小时。 </li><li>诱导并用注射的持续时间期间2.5％的异氟醚麻醉下保持鼠标化。捏鼠标的脚垫来检测反射反应。如果没有检测到，有效的麻醉水平达到了。无菌眼润滑剂，然后应用到防止角膜干燥。 </li><li>加载一个30 G½在“超精细”注射器正是50微升的细胞悬液。确保所有的气泡被从注射器柱通过敲击注射器的侧撞出气泡除去。 </li><li>将鼠标（麻醉下）腹朝上。 </li><li>用酒精棉签清洁注射部位和水平，并直接引入进针第四届乳腺脂肪垫，慢慢地免除注射器体积。 </li><li>用棉签清理任何可能的泄漏。 </li><li>让小鼠从麻醉中恢复。 </li><li>保持丁丙诺啡（0.05毫克/千克）的疼痛管理管理SC，每8小时2天。 </li></ol> 4。在4T1荷瘤BALB / c小鼠管理围手术期处理<ol><li> 13天后肿瘤细胞注射，使用外部卡尺测量原发性肿瘤。测量的最大长径（长度）和最大横直径（宽度）与卡钳。修改后的椭圆体的公式用于计算肿瘤体积（ 肿瘤体积 = 1/2（ 长度为宽度的 2） </li><li> 13天后肿瘤细胞注射，原发肿瘤应该测量大约1 立方厘米 。当这个肿瘤测量达到后，准备围手术期治疗试剂。 </li><li>溶瘤病毒是创新的治疗，人们可以在围手术期管理。制备溶瘤病毒（1×10 9 PFU / 1毫升）在1x无菌PBS中，置于冰上注射前。 </li><li>安全和鼠标的地方为阻挡器尾静脉注射。 </li><li>轻轻地热鼠尾在温暖的自来水，以可视化的侧面尾静脉。 </li><li>恰恰与100微升的溶瘤病毒治疗加载一个27 G½在“胰岛素注射器”。确保所有气泡从注射器列中删除。 </li><li>用1×10 8 PFU/100微升/鼠注入鼠标的侧面尾静脉中的一个。如果针被适当地插入侧脉，无阻力，应按压注射器时，可以感觉到。 </li></ol> 5。原发肿瘤和腹部左侧肾切除术中完整切除<ol><li> 14天后肿瘤细胞注射，开始例行的围手术期护理以下渥太华动物护理和兽医服务的大学认可的协议。皮下注射治疗小鼠丁丙诺啡（0.05毫克/千克）手术疼痛管理前1小时。诱发和使用在手术过程中2.5％异氟烷维持麻醉。 üNDER无菌条件下，使用无菌手术器械，手术部位被剃光，并擦洗。动物被施用在手术前皮下流体和眼的润滑剂。 </li><li>做一个小切口（1-2厘米长）从乳腺脂肪垫轻轻地，彻底切除原发肿瘤4T1。 </li><li>关闭切口用2-39毫米主食。 </li><li>通过沿小鼠的腹侧中线皮肤和皮下组织层切割露出腹部。 </li><li>做一个切口在白线（3-4厘米）来访问小鼠腹膜。 </li><li>通过移动覆盖大便到侧露出腹部的左内侧。确保肠子都保持湿润，用生理盐水浸湿无菌纱布。 </li><li>用钝对手术镊子，轻轻抓住了左肾。 </li><li>使用3-0涂蜡编织丝线绑成一个环，结扎左肾肾门和3外科结固定。与surgi取出左肾卡尔剪刀。 </li><li>仔细检查缝合的领带，以确保有足够的止血的实现。 </li><li>使用5-0编织吸收，然后主食皮肤使用9毫米主食层连续缝合环闭合皮下层。步骤5.2至5.10应该要求10分钟/动物。 </li><li>保持丁丙诺啡（0.05毫克/千克）的疼痛管理管理SC，每8小时2天。 </li></ol> 6。小鼠实施安乐死及加工和肺部肿瘤负担定量<ol><li>在28天后4T1肿瘤注射，根据动物护理和兽医服务在渥​​太华大学协议安乐死小鼠。 </li><li>喷下来鼠标用70％乙醇。 </li><li>使最初的切口，用剪刀只是下方的肋骨。 </li><li>通过沿着鼠标的胸腔腹正中线皮肤和皮下组织层切割露出胸部。 </li><li>通过皮肤和组织每侧到NE使横向切口CK鼠标。 </li><li>通过轻轻抓肺而剪断相差结缔组织上方和下方的肺部解剖出肺。 </li><li>预浸提取的肺中冷PBS以除去残留的血液，然后放置在10％缓冲福尔马林。 </li><li>拍摄每个肺肿瘤转移的评估。权衡肺部肿瘤负荷定量。 </li></ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perioperative+influenza+vaccination+reduces+post-operative+metastatic+disease+by+reversing+surgery-induced+dysfunction+in+natural+killer+cells.">Perioperative influenza vaccination reduces post-operative metastatic disease by reversing surgery-induced dysfunction in natural killer cells.</a> Clin Cancer Res. , (2013).</li></ol>

作者宣称没有竞争的财务权益。

手术切除是治疗患者的局部实体恶性肿瘤的中流砥柱。即使完全切除，许多患者制定一个转移复发并最终死于他们的疾病。术后即刻期间提供了一个理想的环境，形成癌转移，调制，在很大程度上，术后NK细胞的抑制。尽管如此，它仍然是一个治疗窗口，在很大程度上忽略。 目前并没有标准的围手术期的抗癌疗法旨在预防术后转移。目前的挑战是确定安全的和有前途的疗法，将激活NK细胞在围手术期，从而防止建立微转移灶。这些疗法必须严格特点是在临床前动物模型安全性和有效性，然后翻译成精心设计的临床试验。 主要比率制定手术应激的小鼠肿瘤模型的纳莱是继手术，以探讨免疫抑制和转移扩散的机制和评估创新免疫疗法具有潜在的癌症患者接受手术切除原发肿瘤备将来使用。为了实现这个目标，加上手术应激一个4T1小鼠乳腺癌模型的开发。而4T1细胞系是鼠“乳腺癌”，使用该细胞系的原因是自发性转移的重现性，使我们能够评估手术应激的一个现实癌模型的影响。在这种情况下，恶性的实际原点比转移潜力和肿瘤生物学那么重要了。我们的模型的第二关键组分是一种动物外科手术，以接近于人类癌症手术的发展。在这种动物手术应激模型，主要4T1乳腺肿瘤后达到1 立方厘米切除。因为癌症手术在人类患者涉及显著的免疫抑制，在“手术应激”治疗组的开放式腹部切除术是另外进行。因为它与更传统的肿瘤切除术在人类进行比较，一个完整的左侧肾切除术的创伤性是非常类似的，以多种类型的外科治疗实体恶性肿瘤，包括手术治疗结肠直肠癌，卵巢癌，肾癌，胰腺癌，肺癌和食道癌癌症。此外，我们会说，发生围手术期的深刻的生理变化，由于剖腹探查+肾切除术，充分再现了发生下列微创手术对于大多数实体恶性肿瘤绝大多数生理变化。为了控制围手术期因素，可能导致过度手术应激和死亡，麻醉时间和手术，并在手术中保持体温的精确定义。所有这些参数都精确地执行了D在我们的手术协议中定义镜像例行围手术期护理在人类癌症患者。 的围手术期处理的定时是一个额外的关键组成部分的围手术期抢救模型。 4T1荷瘤小鼠先前已经治疗流感疫苗3个方案：新辅助（给出前5天手术），围手术期（在手术当天给出）和围手术期+多剂量（在手术当天发出，其次是2额外的剂量给予5天外）。值得注意的是，疫苗治疗的所有3种模式显著降低肺转移22。然而，流感疫苗的围手术期给药作为单剂量减少转移最有效。总的来说，这些实验中突出的治疗窗窄，在转移过程进行干预眼前的围手术期的重要性。 围手术期使用创新的免疫疗法，如oncoly抽动病毒和疫苗已完全被限制在我们的研究小组。我们证明在第一次新颖的溶瘤ORF和牛痘病毒的围手术期用药可逆转NK细胞抑制手术后的动物模型1。更重要的是，本次救援机体免疫功能的关联与在术后转移的形成减少。在人类研究中，术后肿瘤手术患者减少了NK细胞的细胞毒性和围手术期的OV显着提高NK细胞活性在肿瘤患者1。使用市售的预防性疫苗，我们证明了围手术期的流感疫苗接种显著降低肿瘤转移和改善临床前肿瘤模型中NK细胞的细胞毒作用。在人类的研究，流感疫苗显著增强健康人的捐助者和癌症手术的患者22 NK细胞活性。 许多方法被用来减少CANC呃复发，包括化疗和放疗，但这些疗法通常给予星期前（新辅助）或（佐剂）手术后个月。这方面的研究表明，术后早期是决定长期的肿瘤复发率的关键。因此，在这一关键时期的临床干预免疫疗法的形式可能有大量的长期利益。采用自发肺转移和手术应激小鼠模型我们的研究提供了一个独特的机会去探索新的治疗方法，并确定他们的潜力，以防止围手术期免疫抑制癌症手术的患者，从而也降低癌症的复发率。

手术是在治疗癌症治疗实体瘤的重要组成部分，但尽管完全切除，许多患者制定一个转移复发并最终死于他们的疾病。越来越多地，手术后期间，由于生理应激反应手术，包括血液凝固，生长因子的释放，以及免疫抑制的结果，确认为关键时刻为转移的发展。我们组1,2和其他3-5表明，术后早期是一个独特的敏感时间，形成转移灶。 一个负责手术前转移效应的关键机制是自然杀伤（NK）细胞1-3术后功能障碍。 NK细胞是参与肿瘤的生长和转移6的控制的先天免疫系统的细胞毒性淋巴细胞。 NK细胞功能障碍在手术后已被记录我Ñ ​​人类患者1,7-9和动物模型1,10,11。术后NK细胞抑制水平与第自发和植入转移1,11-14动物模型增加了转移，而在人类的研究，在围手术期低NK细胞活性率较高的癌症复发率和死亡率15,16的关联。 尽管如此，目前没有癌症疗法专门针对发生癌症手术紧随前转移的变化。围手术期指的机会，其中在转移过程进行干预的治疗窗。虽然传统的癌症治疗，如细 ​​胞毒性化疗，被认为太毒被给予患者大手术17恢复，免疫疗法是一种理想的围手术期管理。围手术期使用重组IL-2和IFN-γ已经探索我治疗早期阶段展示他们的潜力，以防止术后NK细胞的抑制和改善无进展生存期18-21临床试验。不幸的是，进一步的发展受到阻碍这个非特异性细胞因子疗法结合大手术17的耐受性。病毒也NK细胞的强效非特异性活化剂。我们的研究曾证明，复制病毒，如新型抗肿瘤溶瘤病毒（OV）和非复制病毒的疫苗，如流感疫苗，术前给药能抑制手术诱发的NK细胞功能障碍和减轻转移性疾病1,22。 本文所描述的手术模式促进了我们参与了肿瘤细胞术后的扩散和生长机制的了解，并允许我们去探索，可以在围手术期给予新的靶向治疗药物。为了实现这一目标，一个动物制定手术应激和自发转移的一叠模型。该模型利用了鼠标的乳腺癌肿瘤（免疫BALB / c - 4T1），它们能自发地从初级乳腺转移到多遥远的地方，尤其是肺部。在第0天，乳腺癌细胞被植入在小鼠乳腺脂肪垫。在第14天肿瘤后植入，在所有的动物中进行了完整切除原发乳腺肿瘤。动物的一个子集接收的腹部切除术的形式附加手术应激。在第28天，在肺肿瘤结节手术强调和比较不手术对照小鼠中分离和量化。在癌症和手术动物模型，正在研究手术对癌转移的影响及创新免疫疗法，包括复制和非复制病毒为基础的免疫刺激剂的围手术期管理的有效性进行测试，以便在第一时间。

<table border="0" cellpadding="0" cellspacing="0" width="647">
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		<col />
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:111px;">Corning Cell-Gro</td>
			<td style="width:153px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:111px;">HyClone</td>
			<td style="width:153px;">SH30396.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:153px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1X sterile Phosphate Buffered Saline (PBS)</td>
			<td style="width:111px;">Corning cell gro</td>
			<td style="width:153px;">21-031-CV</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine</td>
			<td style="width:111px;">Chiron, Guelph</td>
			<td style="width:153px;">RXN309968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isofluorane</td>
			<td style="width:111px;">Baxter Corp</td>
			<td>1001936040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1/2 ultra-fine syringe</td>
			<td style="width:111px;">Terumo</td>
			<td>30 Gauge, SS05M3009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9mm staples</td>
			<td style="width:111px;">Braintree</td>
			<td style="width:153px;">ACS BX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-0 braided absorbable suture</td>
			<td style="width:111px;">Covidien</td>
			<td style="width:153px;">UL-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3-0 wax braided silk suture</td>
			<td style="width:111px;">Covidien</td>
			<td style="width:153px;">S-194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formalin</td>
			<td style="width:111px;">Fisher</td>
			<td style="width:153px;">SF100-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4T1 tumor cells</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:153px;">CRL-2539</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BALB/c</td>
			<td style="width:111px;">Charles Rivers Labs</td>
			<td style="width:153px;">Strain Code:028</td>
			<td></td>
		</tr>
	</tbody>
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a mouse tumor model of surgical stress to explore the mechanisms of postoperative immunosuppression and evaluate novel perioperative immunotherapies

手术切除是大多数癌症患者的基本治疗方法，但手术引起功能障碍，免疫系统，这已被证实与转移性疾病的动物模型中和在癌症患者中的发展。从我们的组和其他临床前工作已经证明先天免疫功能，特别是NK细胞在术后产生深远的抑制，这起着转移的增强发展的重要作用，手术后。相对较少的动物研究和临床试验都集中在特征和逆转癌症手术的不利影响。采用自发转移性肿瘤和手术应激的严格动物模型，对肺转移的发展癌症手术的提升被证明。在这个模型中，4T1乳癌细胞被植入在小鼠乳腺脂肪垫。在第14天肿瘤后植入，完整切除原发乳腺肿瘤是performeD在所有的动物。动物的一个子集接收的腹部切除术的形式附加手术应激。在第28天，肺肿瘤结节进行量化。当免疫治疗给予立即在术前，检测免疫细胞的活化深刻的防止转移的发展在手术之后。而4T1乳腺肿瘤手术模型允许对腹部手术应激对肿瘤转移的影响的仿真，其适用于其他类型的肿瘤需要进行测试。目前的挑战是确定在临床前小鼠模型的安全和前途的免疫疗法，并将其转化为可行的围手术期治疗应给予癌症手术的患者，以防止转移性疾病的复发。

已开发手术应激而导致肺转移的急剧增强的可重复的小鼠模型。在一天28后4T1肿瘤接种（14天后肿瘤切除术+ / - 腹部肾切除术），肺收获和可视化的转移。手术明显增加肺转移的量相比，未处理的小鼠就证明了肺的照片（ 图1A），肺结节的枚举（ 图1B）和肺重量（ 图1C）。复制溶瘤病毒与灭活流感疫苗的术前给药显著抢救癌手术前转移效应（ 图1A-C）。 要确定NK细胞是否起到防止转移后疫苗治疗调解作用，NK细胞是用抗唾液酸-GM-1在肿瘤转移模型药理耗尽。在不存在NK细胞的细胞中，我们观察到的围手术期的免疫疗法的治疗效果的废除（ 图2A和2B）。该数据表明，肿瘤转移切除在我们的手术应激模型主要通过溶瘤病毒和流感疫苗活化NK细胞和随后的NK介导的肿瘤介导的裂解。下面的溶瘤病毒和流感疫苗的围手术期管理，以进一步刻画NK细胞的功能， 体外 NK细胞杀伤进行了评估。简单地说，汇集和整理DX5 + NK细胞从脾中分离手术强调与对照组小鼠。它们共培养4小时用铬标记的YAC-1靶细胞，在不同的效靶比，接着上清液铬释放用γ计数器测定。 NK细胞的细胞毒作用下溶瘤病毒和INFL的围手术期给药的显著手术引起的缺陷以及一个显著复苏的NK细胞杀伤uenza疫苗相比单纯手术（ 图2C和2D）进行了观察。总之，这些结果表明，围手术期NK细胞的抑制可以成功地治疗和转移性疾病减少了新颖的免疫疗法。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51253/51253fig1highres.jpg" src="/files/ftp_upload/51253/51253fig1.jpg" /> 图1。新型抗肿瘤溶瘤病毒和流感疫苗作为对肺转移 4T1肺肿瘤转移时的指示治疗组由代表肺（一）照片，（二）肺 ​​肿瘤枚举28日。评估手术引起的围手术期强化治疗结节和（C）肺湿重。数据代表3次类似的实验，其中n = 5-10/group（*，P = 0.01; **，P &lt;0.0001;<html> NS，不显著）。 <a href="https://www.jove.com/files/ftp_upload/51253/51253fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51253/51253fig2highres.jpg" src="/files/ftp_upload/51253/51253fig2.jpg" /> 图2。手术应激通过削弱NK细胞增加肺肿瘤转移。 （A，B）肺肿瘤转移的定量手术强调与新颖的围手术期治疗治疗的小鼠（C，D）纯化DX5 + NK细胞从手术的能力，强调和未经处理的控制，以杀死肿瘤细胞。数据显示为平均百分比 - 从一式三份孔的指示é铬释放的（+ / SD）：T比率。数据代表3次类似的实验，其中n = 4-5/group（*，P = 0.01; **，P &lt;0.005，NS，不显著）。/ www.jove.com/files/ftp_upload/51253/51253fig2highres.jpg“目标=”_blank“&gt;请点击这里查看该图的放大版本。

Watch this Scientific Journal Video about A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies at JoVE.c

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

手术应激的小鼠肿瘤模型来探讨术后免疫抑制如何促进转移性疾病，并评价免疫围手术期治疗。

手术应激探索术后免疫抑制的机制和评估新围手术期免疫疗法的小鼠肿瘤模型

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

a-mouse-tumor-model-surgical-stress-to-explore-mechanisms

Research

JoVE Journal

Medicine

29.8K 次观看.  Ottawa Hospital Research Institute. 手术应激的小鼠肿瘤模型来探讨术后免疫抑制如何促进转移性疾病，并评价免疫围手术期治疗。 

视频: A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

常温心脏骤停和心肺复苏术：缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型：在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...