The syngeneic rat mesothelioma II-45 cell line was a kind gift from A/Prof. Emanuela Felley-Bosco, Zurich University.

所有涉及动物的程序进行，根据实践中的动物的护理和使用用于科学目的的澳大利亚准则的建议。该协议为这项研究是经皇家北岸医院的动物护理和伦理委员会。女菲舍尔344大鼠（F344，150-200克）保持在卡恩斯设施，Kolling研究所标准条件（12小时亮/暗周期和自由获得食物和水）下。 注意:一个流程图，用于所有实验程序是在图1。 1.准备细胞植入<ol><li>培养大鼠间皮瘤Ⅱ-45细胞系（也称为IL-45;由腹膜引入青石棉的衍生）的RPMI 1640（RPMI）培养基补充有10％胎牛血清（FBS）和生长在标准条件（37° Ç湿润培养箱5％的CO 2）。 Mainta在通过传代和传代培养在约1:50每周两次在75厘米2烧瓶中。 </li><li>制备试剂的细胞培养和温暖的等分试样在37℃。所需试剂包括无血清RPMI培养基（SFM），RPMI有10％FBS，磷酸盐缓冲盐水（PBS）和0.5％胰蛋白酶-EDTA。 </li><li>培养的细胞植入到约70-80％汇合。这保证它们在线性增长阶段。 </li><li>收获细胞通过丢弃媒体，用5ml无菌PBS洗涤一次，然后加入3毫升0.5％胰蛋白酶-EDTA。 </li><li>返回烧瓶培养箱中约23分钟，直到所有的细胞变成非粘附。 </li><li>一旦细胞是非粘附的，加入3 ml的的RPMI有10％FBS以灭活胰蛋白酶。收集和离心细胞，在300×g离心3分钟。 </li><li>在300×g离心再次在10ml SFM和离心机的洗涤细胞沉淀3分钟。 </li><li>洗涤细胞沉淀再次用10ml的SFM的离心如上。 </li><li>悬浮细胞在10ml的SFM，并执行使用血细胞计数器或类似仪器细胞计数。 </li><li>稀释细胞，使100微升含有细胞被植入的量。 注意:肿瘤生长已被证明在一个剂量低至100个细胞在100μl但一个标准剂量是500,000个细胞在100μl。 </li><li>在媒体准备足够细胞被植入大鼠的数量（即，将100μl/鼠），再加上至少0.5 ml的额外以补偿从引发和针的死体积损失。 </li><li>准备足够的SFM（无细胞）为植入对照大鼠（即，将100μl/鼠），再加上至少0.5 ml的额外。 注:细胞和SFM现在准备植入。他们应保持在37℃，并植入2小时内的收获，保持活力。 </li></ol> 2.在体内植入细胞<ol><li>放置F344大鼠（&gt; 13周龄）插入感应腔和麻醉用1.4％异氟烷吸入（或所使用的方法中的设施）。一旦老鼠似乎是睡着了移动它从室到鼻锥（含1.4％异氟醚流），将其放置在其背部与胸部朝上（腹面）。这使内脏解决远离胸腔。请根据机构的协议来保证大鼠reflexes被完全麻醉。 </li><li>剃须正确的区域选择性costalis（胸）区域去除毛发。 </li><li>清洗，用80％V / V乙醇的剃区域。 </li><li>确定注射部位:在右侧，找到第二压盖开始颅。注射部位是0.5厘米近端于此，在从肋笼的尾端的第三和第四肋之间。 （图2A）。 </li><li>轻轻地混合II-45细胞悬浮。慢慢绘制细胞悬浮液（或S​​FM为对照大鼠）到1毫升＃160;注射器没有附加针。如果一个针安装为细胞的图纸上存在对细胞沿着针注入线生长的潜力。附加23G x 1.25针。总理针，并删除任何气泡。 </li><li>一旦注射器和针头都准备，放置20毫米长，直径5毫米间隔件在针轴上。这是用来防止针穿透过深地进入胸膜腔的注射期间。大约5毫米-12毫米暴露针足以通过的肋渗透而不损坏任何器官。 </li><li>慢慢地插入肋骨之间的针，抽回注射器，以确保血管尚未刺破（没血了应该出现在注射器），然后注入100μl的细胞或SFM。 （图2B）。 </li><li>卸下针并轻轻地滚动鼠到另一边蔓延细胞的胸腔。 </li><li>将老鼠进笼子，并检查恢复。钍Ë老鼠应该是在1分钟内清醒并开始走动。 </li><li>重复使用新针头每只大鼠。重复使用相同的针将导致细胞生长沿着针的注入线。 </li><li>每天监视福祉的动物。 </li><li>动物实施安乐死在道义上定义端点作为管辖机构动物伦理委员会。对于大鼠在这些实验伦理终点是重量损失大于10％或呼吸困难。 </li></ol> 3.尾静脉采血<ol><li>如果血液要立即采集后的细胞植入，保持麻醉的老鼠。如果采血在其他时间点，麻醉用异氟醚1.4％的老鼠。请根据机构的协议来保证大鼠reflexes完全麻醉。 </li><li>将大鼠倒向一侧，并找到一个侧尾静脉。 </li><li>消毒用80％乙醇的尾巴和标签0.5ml的EDTAÇollection管。 </li><li>为了收集血液，总是从尾巴的尾端（的方式大约有三分之一沿）。这允许进一步尝试更靠近尾部的颅端的情况下的第一次尝试是不成功的。千万不要重新取样尾部，因为这可能会导致血液凝块。 </li><li>定位一个23G x 1.25针平行于侧静脉和它，所以它渗透约10毫米（图3A）滑入静脉以小角度。 </li><li>注意:如果静脉已成功刺破血液将在针（图3B）的连接端可见。 </li><li>一滴血将形成在尾在针穿刺部位。收集用吸管和转移到标记的0.5毫升（或更小）的EDTA收集管这血。对于免疫细胞测定25微升是足够的。应用纱布的压力，穿刺部位，直到出血停止。 </li><li>弗里克管血混合血液和EDTA公关事件凝固。保持采血并与EDTA作短越好，以防止凝血混合之间的时间。 </li><li>当在室温采集血液从多个大鼠店EDTA-血样在机架中直至分析。在2个小时的采集过程中血液。 </li></ol> 4.样品制备免疫细胞剖析使用基于微珠的方法注意:此单一平台方法依赖于使用具有珠粒每个样品已知数市售绝对计数管中。这些管包含冻干丸粒样品制备过程中溶解，释放珠粒。珠荧光标记和通过门控的珠子群，绝对计数可以计算出来。 <ol><li>确保EDTA全血样品是通过将其放置在一个缓慢旋转混合器几分钟充分混合。标签1的绝对计数管每个样品。一份载有珠颗粒应为t下方可见在管的底部他金属珠保持器。 </li><li>转移25微升EDTA全血成标记的绝对计数管。珠颗粒会溶解在加血。 </li><li>向每个试管添加20微升抗大鼠T / B /天然杀伤（NK）细胞的鸡尾酒，将10μl抗大鼠CD8A的PE，10微升抗大鼠CD4（域1）的FITC和10μl抗大鼠的CD45 PE / Cy7的（图4A）。荧光团在表1中所定义。 </li><li>离心反应管短暂地（300×g离心），以确保所述的抗体和细胞在管的底部，而不是粘在管的一侧。涡旋混合并孵育15分钟，在RT。 </li><li>以裂解红血细胞中添加的10毫摩尔Tris 400微升，0.15M氯化铵缓冲液（pH 7.5）和涡旋以混合。裂解完成时，样品出现半透明的，不混浊（ 图4B和C）。未溶解的样品完全会导致增加研发背景和当流动分析仪假性上升计数。 </li></ol>样品5.流式细胞仪处理注意:请在四色流式细胞仪。 <ol><li>打开采集模式和与8描述一个新的模板的软件，如图5所描绘。 </li><li>调整仪器设置为表1中列出并设置栅极R1（FITC [FL - 1] 与 APC [FL - 4]）， 图5AI）计数荧光珠。其他的门都没有在此次收购阶段一样重要，但将需要进行分析。在这个协议中使用的绝对计数的珠含有荧光染料，可在任何通道被检测到，虽然是最弱的蓝色通道。 </li><li>使用准备好的控制血液样品，涡旋，然后加载到流式细胞仪和以低速（12微升/分钟）上设置模式运行，以便数据采集门可以调整。 </li><li>将收购聚集在R1珠门10,000个事件。 </li><li>建立一个文件夹来记录数据，并设置文件号和标签示例文件中采集菜单。 </li><li>加载待分析样品到流式细胞仪，设置流速介质（35微升/分钟）。运行在相同的流速各样品中。流速，可能需要改变，以低（12微升/分钟）或高（60微升/分钟），但介质一般是合适的。以这样的速度，大约需要90至120秒，以获得万珠事件每个样品。 </li><li>一旦样品被加载观看散点图以确保事件出现在R 1胎圈栅极。最初可存在于样品压力引起的漂移在散点图有些不稳定。等待这个稳定。 </li><li>一旦稳定下来，点击获取，并允许样品运行。一旦流式细胞仪已完成收购R1万珠事件流式细胞仪将停止采集并保存所有的数据。 </li><li>取下样品和废弃流涂可以。细胞仪现在准备下一个样品。运行所有的样品，然后进行分析模式。 </li></ol> 6.免疫细胞分析注意:门控策略和布尔代数被用来定义每个细胞群。布尔代数是一个基于逻辑分析方法，其允许在一个单一的定义多个操作。流式细胞仪的分析软件（例如，BD CELLQUEST）允许采用布尔代数。该公式用于积极地占显着的负反应性，其有助于限定细胞更具体标识每个细胞群。 &#39;区域&#39;被用来定义一个"门"。区域限定一个2维空间，而栅极可以通过代数运算符连接众多的区域组成（+，* - ， 在表2中定义）。 <ol><li>切换软件来分析模式。分析模板应产生匹配<str翁&gt;图5示出与情节和大门。 </li><li>分析每个单独的文件（ 即，每个样品）分开。设置栅极R1至到R9和然后设置算法对于每个细胞类型如表2中所定义（也示于图5）。 </li><li>使用电池的统计数据计数器来计算通过门和算法（ 表2和图5）中定义的单个细胞群。该算法将在小区统计计数器自动调节细胞的数量。 </li><li>使用下列公式计算细胞亚群: <img alt="式（1）" src="/files/ftp_upload/53019/53019eq1.jpg" /> 注意:所计数的细胞数的事件（例如，CD4 + T细胞的事件）被利用上述方程，得到每微升血液细胞数计数。例子示于图5。 </li></ol>

<ol>
	<li>Kao, S. C., 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=Malignant+mesothelioma.">Malignant mesothelioma.</a> Intern Med J. 40 (11), 742-7450 (2010).</li><li>Zucali, P. A., 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=Advances+in+the+biology+of+malignant+pleural+mesothelioma.">Advances in the biology of malignant pleural mesothelioma.</a> Cancer Treat Rev. 37 (7), 543-558 (2011).</li><li>Olsen, N. J., 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=Increasing+incidence+of+malignant+mesothelioma+after+exposure+to+asbestos+during+home+maintenance+and+renovation.">Increasing incidence of malignant mesothelioma after exposure to asbestos during home maintenance and renovation.</a> Med J Aust. 195 (5), 271-274 (2011).</li><li>Zucali, P. A., 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=Thymidylate+synthase+and+excision+repair+cross-complementing+group-1+as+predictors+of+responsiveness+in+mesothelioma+patients+treated+with+pemetrexed/carboplatin.">Thymidylate synthase and excision repair cross-complementing group-1 as predictors of responsiveness in mesothelioma patients treated with pemetrexed/carboplatin.</a> Clin Cancer Res. 17 (8), 2581-2590 (2011).</li><li>Vogelzang, N. J., 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=Phase+III+study+of+pemetrexed+in+combination+with+cisplatin+versus+cisplatin+alone+in+patients+with+malignant+pleural+mesothelioma.">Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma.</a> J Clin Oncol. 21 (14), 2636-2644 (2003).</li><li>Lowenstein, P. R., Castro, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncertainty+in+the+translation+of+preclinical+experiments+to+clinical+trials.+Why+do+most+phase+III+clinical+trials+fail?.">Uncertainty in the translation of preclinical experiments to clinical trials. Why do most phase III clinical trials fail?.</a> Curr Gene Ther. 9 (5), 368-374 (2009).</li><li>Kamb, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+wrong+with+our+cancer+models.">What's wrong with our cancer models.</a> Nat Rev Drug Discov. 4 (2), 161-165 (2005).</li><li>Yakisich, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Algorithm+for+the+Preclinical+Screening+of+Anticancer+Drugs+Effective+against+Brain+Tumors.">An Algorithm for the Preclinical Screening of Anticancer Drugs Effective against Brain Tumors.</a> ISRN Pharmacol. 2012, 513580 (2012).</li><li>Basu, D., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+microenvironments+within+mouse+models+that+enhance+tumor+aggressiveness.">Defining microenvironments within mouse models that enhance tumor aggressiveness.</a> Cancer Biol Ther. 8 (4), 380-381 (2009).</li><li>Abolhassani, M., 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=Screening+of+well-established+drugs+targeting+cancer+metabolism:+reproducibility+of+the+efficacy+of+a+highly+effective+drug+combination+in+mice.">Screening of well-established drugs targeting cancer metabolism: reproducibility of the efficacy of a highly effective drug combination in mice.</a> Invest New Drugs. 4 (4), 1331-1342 (2011).</li><li>Hudson, A. L., 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=Establishing+a+panel+of+chemo-resistant+mesothelioma+models+for+investigating+chemo-resistance+and+identifying+new+treatments+for+mesothelioma.">Establishing a panel of chemo-resistant mesothelioma models for investigating chemo-resistance and identifying new treatments for mesothelioma.</a> Sci Rep. 4, 6152 (2014).</li><li>Iannaccone, P. M., Jacob, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats!.">Rats!.</a> Dis Model Mech. 2 (5-6), 206-210 (2009).</li><li>Craighead, J. E., 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=Characteristics+of+tumors+and+tumor+cells+cultured+from+experimental+asbestos-induced+mesotheliomas+in+rats.">Characteristics of tumors and tumor cells cultured from experimental asbestos-induced mesotheliomas in rats.</a> Am J Pathol. 129 (3), 448-462 (1987).</li><li>Hunter, S. D., 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=Lymphocyte+subset+analysis+by+Boolean+algebra:+a+phenotypic+approach+using+a+cocktail+of+5+antibodies+and+3+color+immunofluorescence.">Lymphocyte subset analysis by Boolean algebra: a phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence.</a> Cytometry. 15 (3), 258-266 (1994).</li><li>Brando, B., 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=Cytofluorometric+methods+for+assessing+absolute+numbers+of+cell+subsets+in+blood.+European+Working+Group+on+Clinical+Cell+Analysis.">Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.</a> Cytometry. 42 (6), 327-346 (2000).</li><li>Schnizlein-Bick, C. T., 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=Evaluation+of+TruCount+absolute-count+tubes+for+determining+CD4+and+CD8+cell+numbers+in+human+immunodeficiency+virus-positive+adults.">Evaluation of TruCount absolute-count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus-positive adults.</a> Clin Diagn Lab Immunol. 7 (3), 336-343 (2000).</li><li>Gajkowska, A., 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=Flow+cytometric+enumeration+of+CD34++hematopoietic+stem+and+progenitor+cells+in+leukapheresis+product+and+bone+marrow+for+clinical+transplantation:+a+comparison+of+three+methods.">Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods.</a> Folia Histochem Cytobiol. 44 (1), 53-60 (2006).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Kao, S. C., 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=High+blood+neutrophil-to-lymphocyte+ratio+is+an+indicator+of+poor+prognosis+in+malignant+mesothelioma+patients+undergoing+systemic+therapy.">High blood neutrophil-to-lymphocyte ratio is an indicator of poor prognosis in malignant mesothelioma patients undergoing systemic therapy.</a> Clin Cancer Res. 16 (23), 5805-5813 (2010).</li><li>Kao, S. C., 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=Validation+of+prognostic+factors+in+malignant+pleural+mesothelioma:+a+retrospective+analysis+of+data+from+patients+seeking+compensation+from+the+New+South+Wales+dust+diseases+board.">Validation of prognostic factors in malignant pleural mesothelioma: a retrospective analysis of data from patients seeking compensation from the New South Wales dust diseases board.</a> Clin Lung Cancer. 14 (1), 70-77 (2013).</li><li>Burt, B. M., 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=Circulating+and+tumor-infiltrating+myeloid+cells+predict+survival+in+human+pleural+mesothelioma.">Circulating and tumor-infiltrating myeloid cells predict survival in human pleural mesothelioma.</a> Cancer. 117 (22), 5234-5244 (2011).</li><li>Weir, C., 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=Streptavidin:+a+novel+immunostimulant+for+the+selection+and+delivery+of+autologous+and+syngeneic+tumor+vaccines.">Streptavidin: a novel immunostimulant for the selection and delivery of autologous and syngeneic tumor vaccines.</a> Cancer Immunol Res. 2 (5), 469-479 (2014).</li></ol>

The authors have nothing to disclose.

本文详述了胸膜间皮瘤大鼠的同源原位模型以及用于通过纵向血液取样监测疾病进展的简单方法的产生的方法。 在II-45模型通过将菲舍尔344大鼠石棉纤维13开发的。虽然这种曝光表示主机石棉免疫系统的相互作用的真实动态间皮瘤发病机制，它具有长的滞后时间（以年来产生），并可能是危险的，研究人员由于可能暴露于石棉纤维。 Ⅱ-45细胞的原位移植进入胸膜动物腔提供癌症发展一种安全和快速模型模仿人类疾病中的免疫能力的主机 11。但是确切的方法和原则，坚持在这些实验过程没有明确定义。该过程在一个高度可再生的模型，该模型是RELA描述的结果tively独立的操作员的经验。关键步骤是1，收获的II-45的细胞在指数生长期和活细胞计数准确; 2，植入部位的正确定位，确保针头不会刺入太深和穿刺肺或损害心脏，和3，维护和监控福祉的动物。 的Ⅱ-45细胞系生长速度快，并且需要传代，每3至4天的培养，因此准备用于植入时，细胞应该是亚培养和日生长监测。细胞应收获并计数在指数生长期时， 即，在大约 70％汇合。细胞的无血清培养基，而不是PBS的再悬浮减少压力对细胞的同时计数，准备植入。准确细胞计数再现性是至关重要的，特别是当与低细胞数目（例如，100〜1000）给药。在SUS养老金应是均匀的，并且由单个细胞（无团块）的。 100微升注入量能够准确的计量，而无需通过过多的液体在胸膜腔impairing肺扩张。在植入的关键步骤是正确的定位，并限制该针穿透至不超过12毫米的深度。针在注射部位的正确定位是非常重要的，以确保将细胞植入到胸膜腔和不进入腹膜腔。错误地植入细胞过低可能会导致间皮瘤的肿瘤通过隔膜越来越大，进入肝脏和腹腔。限制所述穿透深度的针也是极为重要的，以防止死亡率的内部器官损伤的结果。这种限制可以通过使用间隔物的在一个长针（如图所示），或者使用短针与5mm的轴长12毫米而实现。在我们的手中，无不良事件发生在细胞植入。同样重要的是每一个植入后更换针头。如果不这样做可能会导致肿瘤生长出胸腔内沿注射线。胸膜腔的外面这种增长是由于残余细胞上的针轴，并可能导致在延长的存活。 在我们手中这款机型具有100％的肿瘤细胞植入率时给予≥1×10 3细胞 。在II-45间皮瘤模型疾病进展相当快，为少于40天，当1×10 4个细胞植入和少于20天的5×10 5个细胞的剂量。这可能会限制该模型由于某种疗法的临床前试验的有用性。在II-45细胞，而不是市售可从始发13来采购的许可。然而，同样的植入技术和原理也可以应用于其它的大鼠间皮瘤细胞可得自细胞系回购系itories当与相关同基因大鼠品系匹配。最优细胞剂量可以与细胞系改变，并且将需要为每一行确定。 监测疾病进展和福利动物是困难的，因为在肿瘤内部的，不能由大小监测。因此，我们寻求开发监测，可以预测其条件恶化的动物的方法，如增加NLR或免疫细胞标记物的其他变化前躯体症状变得可见。开发的血液屏幕只使用25微升末梢血，这样经常性抽样能够以最小的痛苦的动物进行。所描述的采样技术需要实践来获得必要的专业知识。然而，一旦掌握了它的快​​速和微创。 在血液分析的关键步骤是防止血液样品凝血通过快速传输到EDTA管中。凝血样本应予舍弃的计数将是不准确的。另一个关键步骤是前细胞分析红细胞裂解。如果红色细胞没有充分溶解，增加的背景将错误地升高计数。虽然点图和门控的初始设置非常耗时，一旦设置，模板保存，该法只需要很少的调整。所用的门控策略使用多参数布尔代数的方法来解卷积的数据。通过使用排他性已知的组合和共享抗原的算法用于栅战略的每条线在该六维数据矩阵14限定离散的细胞类型。这加上比率量度基于珠子的技术15-17的建立准确性具有的优点的有利的任何定义的细胞类型的枚举作为细胞两者的比例和每微升的绝对计数。 人们越来越认识到，免疫的System p规定在癌症18的发病机制中起重要作用。两对免疫系统未能识别和破坏肿瘤细胞和免疫细胞的肿瘤抑制可以导致不受控制的肿瘤生长。因此，夹杂物的免疫能力的肿瘤微环境是一个重要的考虑因素建模癌症，并且是同基因癌症模型的一个主要优点时。这些免疫能力的模型创建的肿瘤生长和免疫相互作用，这是缺乏异种模型逼真和临床相关的环境。 在这项研究中伴随疾病的进展， 即，淋巴细胞计数，单核细胞计数与NLR鉴定免疫参数，也被证实与预后人类间皮瘤19-21提供的证据，该模型的相关性差，血液检查。在介绍的模型中，监测免疫指标的临床应用增加智慧小时较长时间场（低剂量），并且可以进一步通过更频繁的采样在稍后该疾病的阶段提高。虽然本文的重点上间皮瘤的模型，并监控通过外周血取样肿瘤进展的能力可以被用于所有的同基因癌症模型。此外，分析还可以用于评估不同的治疗的免疫学应答。以前，我们已经使用此测定法来监测在一个同基因胶质瘤模型（9L）22到抗癌疫苗疗法的响应。在这项研究中，CD4和CD8 T细胞是最信息标记。我们也用这种试验和II-45间皮瘤模型来识别在肿瘤抵抗不同化学治疗剂11的免疫应答的差异。在这项研究中，除了CD8 T细胞的所有参数表现出明显的差异。在这里所描述的疾病进展的纵向监测强调了不同的免疫细胞参数潜力心病从事癌症同源原位模型大鼠时，涉及与癌症的发展和福祉的动物。

恶性间皮瘤（MM）是一种侵袭性的恶性肿瘤其来自转化的细胞中的膜（间皮），该系肺和腹腔，心脏和内部繁殖器官和是肺腔或胸膜1,2的最常见的原发性肿瘤。暴露于石棉纤维占80％，所有的MM，虽然对石棉的使用禁令推出几十年前，在大多数西方国家，其社会各界的广泛使用已经留下了一个致命的遗产。据世界卫生组织估计，107,000名员工每年死于与石棉有关的疾病，与死亡率持续增加。一种新的非职业入射波也正在和有何时，在什么水平，这将达到峰值3知之甚少。 大多数人的MM被确诊晚了，当全身化疗代表的唯一可行的选择4之一。最effecti已经化疗（顺铂5培美曲塞在一起）当前的"护理标准"被认定在10年前。但是失败这种治疗是不可避免的，也没有成熟的第二行选项，使患者的一个严峻的预后只有12 个月2，中位生存期。因此，目前迫切未满足的需要更有效的治疗方法。尽管一批临床试验中的新疗法的检查没有导致的变化在实际中。这是部分由于低5％转移的临床前的结果，一般在异种移植小鼠模型中进行，给临床设定6-8。这类模型不忠实概括在非生理位置中发生的肿瘤微环境的复杂的方面，经常在没有免疫系统功能9。 同源原位模型创建比C一显著更现实的肿瘤环境ommonly使用皮下异种移植模型作为出现在正确的生理部位有一个完整的免疫系统10,11的肿瘤。的大鼠的尺寸较大增强其作为一种啮齿动物疾病模型的使用，尤其是在药物研究，其中串行抽血需要来评估治疗反应和毒性12。此外，在模型中，监测疾病进展是困难的，因为在肿瘤（如在胸膜腔）的位置，用在血液循环中发现的因素是非常有吸引力的，监测疾病进展的能力。使用免疫能干大鼠胸膜间​​皮瘤的同基因原位模型的生成进行说明。此外，一种简单的和相对非侵入性的方法，通过测量循环免疫细胞监测胸膜疾病进展还描述。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EDTA Collection tube (0.5ml)</td>
			<td>Greiner Bio One GmbH</td>
			<td>450480</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat T/B/NK Cell cocktail</td>
			<td>BD Pharmingen</td>
			<td>558509</td>
			<td>anti-Rat CD3&#160; APC (IgM clone 1F4), anti-Rat CD45RA Fitc (IgG1 clone OX-33), anti-Rat CD161a PE (IgG1 Clone 10/78)</td>
		</tr>
		<tr>
			<td>anti-RAT CD8a PE&#160;</td>
			<td>Biolegend</td>
			<td>200608</td>
			<td>(IgG1vClone G28)</td>
		</tr>
		<tr>
			<td>anti-Rat CD4 FITC (Domain 1)&#160;</td>
			<td>Biolegend</td>
			<td>203406</td>
			<td>(IgG1 Clone OX-38)</td>
		</tr>
		<tr>
			<td>anti-Rat CD45 PE/Cy7&#160;</td>
			<td>Biolegend</td>
			<td>202214</td>
			<td>(IgG1 Clone OX-1)</td>
		</tr>
		<tr>
			<td>TruCount&#8482; Tubes</td>
			<td>Becton Dickinson</td>
			<td>340334</td>
			<td>Box of 50 absolute counting tubes</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>foetal bovine serum (FBS)</td>
			<td>Scientifix</td>
			<td>FBS500-S (lot# 010101-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Medicago AB</td>
			<td>09-9400-100</td>
			<td></td>
		</tr>
		<tr>
			<td>23Gx1&#188; Needle</td>
			<td>Becton Dickinson</td>
			<td>302008</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>302 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fischer 344 Rat</td>
			<td>Animal Resources Centre, Perth Australia</td>
			<td>F344</td>
			<td></td>
		</tr>
		<tr>
			<td>I.S.O (Isoflurane USP)</td>
			<td>Veterinary Companys Australia (VCA)&#160;</td>
			<td>B7058</td>
			<td></td>
		</tr>
		<tr>
			<td>II-45 Rat Mesothelioma line</td>
			<td>Zurich University</td>
			<td></td>
			<td>Note: The cell line was given as a gift and is not commercially available at the ATCC</td>
		</tr>
		<tr>
			<td>FACSCalibur&#8482; 4 colour</td>
			<td>Becton Dickinson</td>
			<td>342975</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-HCL</td>
			<td>SIGMA</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>SIGMA</td>
			<td>9718</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthetic Machine (The stinger)</td>
			<td>Advanced Anaesthesia specialists</td>
			<td>#00449</td>
			<td></td>
		</tr>
	</tbody>
</table>

orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model

在免疫为基础的治疗的癌症如疫苗和免疫检查点抑制剂，和在治疗响应肿瘤微环境中的作用的进一步了解感兴趣的巨大热潮，共同指向需要免疫能干正位模型的临床前试验这些新的治疗方法。本文演示了如何建立胸膜恶性间皮瘤原位免疫能力的动物模型。在原位模型监测疾病进展是由肿瘤的内部位置混淆。纵向监测疾病的进展及其对循环免疫细胞在这方面和癌症等大鼠模型的效果，单管流式细胞术检测只需要25全血中描述微升。这提供了七个免疫指标准确定量:总淋巴细胞，单核细胞和嗜中性粒细胞，以及T细胞亚群CD4和CD8，B细胞和自然杀伤细胞。不同的潜艇这些参数的ETS在不同的情况和模型有用，与中性粒细胞淋巴细胞比率具有用于监测疾病进程中的间皮瘤模型最大的效用。分析循环使用这种单管方法还可以协助监测响应免疫为基础的治疗和了解，导致治疗成功或失败的根本机制免疫细胞的水平。

在本文用于使用II-45细胞胸膜间皮瘤的原位模型​​的生成的方法，导致动物屈服于间皮瘤可再现的和快速的时间内，没有大鼠垂死由于植入方法。细胞植入的数量的滴定中确定1×10 3个细胞是需要的充分渗透模型（100％的植入）的最小数目。的不同数目的细胞植入到大鼠的改变疾病的时间过程中没有出现影响疾病的严重程度。动物接受1×10 3，1×10 4和5×10 5个细胞40天，30和20天（图6）达到道德定义端点。 血液从老鼠大约每星期收集两次，无并发症观察相关的集合。要注意的是不超过10％的总血量的是很重要的（或大约2毫升）必须被收集在一个2周。这门控相结合的战略和布尔代数的设立是为了识别淋巴细胞，单核细胞和嗜中性粒细胞和淋巴细胞亚群T，CD4 + T，CD8 + T，B细胞和NK细胞流式细胞仪检测。的平均值和范围对于每个细胞类型，使用控制鼠（n = 5），并进行比较的II-45植入大鼠在端点的平均值和范围（表3）成立。首先比较端点值，以确定差异是否存在健康和患病动物之间的免疫细胞计数。相较于健康对照大鼠，总淋巴细胞，CD4 + T细胞，B淋巴细胞和NK细胞中的II-45植入大鼠端点降低而单核细胞，中性粒细胞和嗜中性粒细胞向淋巴细胞比率（NLR）增加。 要确定是否这些免疫细胞参数也改变与疾病进展，因此，可作为替代标志˚F或监测疾病，纵向的样品也进行了比较（图7）。最翔实纵向参数是NLR与检测出最高至7天的伦理端点（图7A）之前增加。对于大鼠大剂量的细胞，7天及12天植入后的检测到高NLRS，而对于大鼠低剂量的细胞中，NLR使用了18天，22总淋巴细胞计数的增加与疾病进展降低大鼠一较长的病日久模式（低细胞剂量:1×10 4， 图7B）。这种减少并不明显大鼠具有快速时间过程（高细胞剂量:5×5）。单核细胞维持在正常水平，直到终端集合时严重增加计数经常观察（图7C）。的其它免疫细胞计数（中性粒细胞，NK细胞，B细胞和CD4和CD8 T细胞）更变量，从而减少信息量。 图1.流程图的实验过程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53019/53019fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53019/53019fig2.jpg" /> 图2.地点与本站植入。首先将大鼠麻醉，置于在它的后面和右区域选择性costalis（胸部）区域被剃以除去皮毛。在右侧，从顶部起第二乳头被识别和注射部位位于0.5厘米近端于此，在从胸廓（A）的底部的第3次和第4次肋之间。的隔离物，然后放置在所制备的针头和注射器，以防止针穿透过深地进入胸膜<html>腔体和100微升细胞或SFM的注入（B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53019/53019fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53019/53019fig3.jpg" /> 图3.采样技术采集血液的尾巴。放置一个23G x 1.25针平行于侧脉（A）。滑动针头插入静脉以小角度，大约10毫米深，并保持有几秒钟，直到血液是可见的（B），然后卸下针头。用吸管（C）收集的血液。如果有必要轻轻抚摩尾静脉，以保证血液继续流动。移液器血液进入0.5ml的EDTA收集管（D），搅拌，当您去，以防止凝血。<html> arge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53019/53019fig4.jpg" /> 图4.标签中的绝对数量管流式细胞仪分析血液样品的血 （25微升）和抗体被加入到绝对计数管（A）中 。然后将管涡旋并温育15分钟，在室温下进行。前加入氯化铵缓冲液以裂解红血细胞，所述样品出现混浊或不透明（B）中 。一旦溶解，样品出现半透明的，并准备流式细胞仪分析（C）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53019/53019fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ES / ftp_upload / 53019 / 53019fig5.jpg"/&gt; 图5.门控策略进行流式细胞术分析。甲6维数据集可以识别淋巴细胞，单核细胞和嗜中性粒细胞和淋巴细胞亚群T，CD4 + T，CD8 + T，B和NK细胞。所定义的门控策略和算法二者结合的阴性和阳性反应性定义每个小区类型允许特定标识。在每个绝对计数管（在袋的每个管中找到）小珠的数量是记录用于每一样本和所需的计算，如图每微升血液细胞对于每个子集的数量。完整的选通算法对每个小区子集被列出的分析曲线的右侧。（艾）珠（R 1）由FITC（FL-1）与APC（FL-4）最初计数。采集设定为约10000个事件（9960在这个例子中）。（AII）碎片（R2）被由FSC从所有后续克确定与 SSC和除去阿婷。（Bi）的在除去碎片（R 2）的FSC 相对于 SSC区分白血细胞种群成淋巴细胞（R 3），单核细胞（R 4）和嗜中性粒细胞（R 5）。（BII）的 （Bi）的确定的细胞群使用证实在常见的白细胞抗原CD45门上的PE / Cy7的（FL-3）与SSC（BIII）的淋巴细胞群（R3）进一步分化成使用CD3门上的APC（FL-4）与SSC T细胞（R6）。（ BIV）的CD8 T细胞是分化为T细胞（R 6）也表达CD8A（R 9）和被选通在PE（FL-2）与SSC淋巴细胞（R 3）。（次）B细胞和NK细胞被定义为淋巴细胞（ R 3），它们是CD3阴性。被B细胞（R 7）从NK细胞分化为它们表达CD45RA和由APC（FL-4）被选通与 FITC（FL-1）。（CII）的CD4 T细胞被定义为淋巴细胞（R 3），该共表达的CD3 （T细胞，R 6）和的CD4（R 8），但不表达任何使用的其它标记。他们使用的是FITC（FL-1）与PE（FL-2）选通区分。 NK细胞被定义为尚未定义的任何剩余的细胞也表达CD161a。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53019/53019fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53019/53019fig6.jpg" /> 图6.存活曲线老鼠注入不同剂量的II-45间皮瘤细胞。大鼠组分别pleurally植入5×10 5（N = 6），1×10 4（N = 2），1×10 3（ n = 2时）或1×10 2（4例）的II-45间皮瘤细胞在100μL的体积，然后监测，直至当它们安乐死伦理端点。使用对数秩（曼特尔 - 考克斯）Gehan-布瑞斯罗夫，魏氏法生存作图。 </<html> P&gt; <img alt="图7" src="/files/ftp_upload/53019/53019fig7.jpg" /> 图7.纵向从大鼠相对均值和范围对于健康对照大鼠间皮瘤循环免疫细胞计数的健康对照意味着针对每个小区类型被示为水平不间断黑线（值显示上的右Y轴每幅图）和范围由灰色阴影区域示出。 Y轴显示每微升血液的计数在规定的细胞类型，或用于NLR值。 X轴是时间过程疾病进展，其中0天是植入的天。纵向结果2只大鼠植入1×10 4个细胞（A，B）和2只大鼠植入5×10 5个细胞（C，D）被示出。（A）中NLR，（B）的总淋巴细胞计数，（C）的单核细胞计数。ge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>探测器</td><td>荧光基团</td><td>详细信息</td><td>电压</td><td>状态</td></tr><tr><td> FSC </td><td> --- </td><td> --- </td><td> E00 </td><td>线性</td></tr><tr><td> SSC </td><td> --- </td><td> --- </td><td> 450 </td><td>线性</td></tr><tr><td> FL-1 </td><td> FITC </td><td>三十零分之五百三十三纳米</td><td> 670 </td><td>登陆</td></tr><tr><td> FL-2 </td><td> PE </td><td>四十零分之五百八十五纳米</td><td> 675 </td><td>登陆</td></tr><tr><td> FL-3 </td><td> PE / Cy7的</td><td> 670nm处（长传） </td><td> 600 </td><td>登陆</td></tr><tr><td> FL-4 </td><td> APC </td><td>二十五分之六百七十五纳米</td><td> 735 </td><td>登陆</td></tr></tbody></table> 表1:用于流CYTO电压检测器设置使用4色流式细胞仪度量数据采集。振幅增益设置为1的所有探测器。 FSC:前向散射，SSC:边撒，FITC:荧光isothyocyanante，PE:藻红蛋白，APC:Allophycocyanine。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>细胞亚群</td><td>细胞标记物</td><td>门控算法</td></tr><tr><td>珠</td><td></td><td> R1 </td></tr><tr><td>碎片</td><td></td><td> R2 </td></tr><tr><td>细胞（泛白细胞） </td><td> CD45 + </td><td> -R2 </td></tr><tr><td>淋巴细胞</td><td> CD45 + FSC与SSC </td><td> R3 * -R2 </td></tr><tr><td>单核细胞</td><td> CD45 +与FSC SSC </td><td> R4 * -R2 </td></tr><tr><td>中性粒细胞</td><td> CD45 + FSC与SSC </td><td> R5 * -R2 </td></tr><tr><td> T细胞（总） </td><td> CD45 + / CD3 + </td><td> R6 * R3 * -R2 </td></tr><tr><td> CD8阳性T CELLS </td><td> CD45 + / CD3 + / CD8A + </td><td> R9 * R6 * R3 * -R2 </td></tr><tr><td> B细胞</td><td> CD45 + / CD3- / CD45RA + </td><td> R7 * R3 * -R2 </td></tr><tr><td> CD4阳性T细胞</td><td> CD45 + / CD3 + / CD4 + </td><td> R 8 * R 6 * R 3 * - （R 7 * R3 * -R2） </td></tr><tr><td>自然杀伤细胞</td><td> CD45 + / CD3- / CD161a + </td><td> R 3 * - [（R 2 + R6）+（R 7 * R3 * -R2）] </td></tr></tbody></table> 表2:细胞标记物和选通算法来表征每个免疫细胞亚群对于门控算法，的"+"符号是指"或"并允许添加两个单独的群一起即使他们的反应不重叠。在" - "符号的意思是"不"，并可以从其他特定人群或先前描述的人口反向内减去一个群体。所述&#39;*&#39;符号的意思是"和"，并限定一个空间，仅开放两个反应重叠。 </P&gt; <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>免疫细胞亚群</td><td colspan="2">控制鼠（n = 5） </td><td colspan="2">在端点Ⅱ-45植入鼠（n = 4） </td></tr><tr><td></td><td>意味着</td><td>范围</td><td>意味着</td><td>范围</td></tr><tr><td>总淋巴细胞</td><td> 165.7 </td><td> 111.5-207.0 </td><td> 97.9 </td><td> 54.4-137.0 </td></tr><tr><td> CD4 + T淋巴细胞</td><td> 72.9 </td><td> 45.4-111.4 </td><td> 38.4 </td><td> 25.6-50.0 </td></tr><tr><td> CD8 + T淋巴细胞</td><td> 36.6 </td><td> 26.0-48.3 </td><td> 32.1 </td><td> 16.1-46 </td></tr><tr><td> B淋巴细胞</td><td> 56.6 </td><td> 36.0-83.9 </td><td> 24.7 </td><td> 11.2-36.0 </td></tr><tr><td> NK细胞</td><td> 9.3 </td><td> 3.1-15.7 </td><td> 4 </td><td> 1.34-7.7 </td></tr><tr><td>单核细胞</td><td> 27.1 </td><td> 12.7-39.0 </td><td> 373。3 </td><td> 42.4-575.8 </td></tr><tr><td>中性粒细胞</td><td> 51.4 </td><td> 24.3-87.0 </td><td> 159.2 </td><td> 51.1-366.0 </td></tr><tr><td> NLR </td><td> 0.3 </td><td> 0.18-0.42 </td><td> 1.8 </td><td> 0.94-2.66 </td></tr></tbody></table> 表3:在健康对照大鼠的均值和免疫细胞的范围（N = 5）和II-45植入大鼠端点（n = 4时）每微升血液结果示。

Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

II-45同基因大鼠间皮瘤模型中的原位植入和外周免疫细胞监测|医学|视频

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

胸膜恶性间皮瘤由II-45间皮瘤细胞进入胸膜免疫活性大鼠腔植入的原位大鼠模型的产生提出。甲流式细胞法分析从25微升血样在这些动物7免疫细胞亚群也被描述。

在II-45同基因大鼠模型间皮瘤原位移植和外周免疫细胞监视

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

orthotopic-implantation-peripheral-immune-cell-monitoring-ii-45

Nanyang Technological University

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9.1K 次观看.  Royal North Shore Hospital, University of Sydney. 胸膜恶性间皮瘤由II-45间皮瘤细胞进入胸膜免疫活性大鼠腔植入的原位大鼠模型的产生提出。甲流式细胞法分析从25微升血样在这些动物7免疫细胞亚群也被描述。 

视频: Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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医学

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

原位Xenografting人类荧光素酶标记的恶性外周神经鞘瘤细胞在体内测试候选治疗药物

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

The Polyvinyl Alcohol Sponge Model Implantation

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing studies are most comprehensive when carried out in vivo. There are many in vivo models available to study acute wound healing, including incisional, excisional, dead space, and burns. Dead space models are artificial, porous implants which are used to study tissue formation and the effects of substances on the wound. Some of the commonly used dead space models include polyvinyl alcohol (PVA) sponges, steel wire mesh cylinders, expanded polytetrafluoroethylene (ePTFE) material, and the Cellstick1,2.
Each dead space model has its own limitations based on its material's composition and implantation methods. The steel wire mesh cylinder model has a lag phase of infiltration after implantation and requires a long amount of time before granulation tissue formation begins1. Later stages of wound healing are best analyzed using the ePTFE model1,4. The Cellstick is a cellulose sponge inside a silicon tube model which is typically used for studying human surgery wounds and wound fluid2. The PVA sponge is limited to acute studies because with time it begins to provoke a foreign body response which causes a giant cell reaction in the animal5. Unlike other materials, PVA sponges are easy to insert and remove, made of inert and non-biodegradable materials and yet are soft enough to be sectioned for histological analysis2,5.
In wound healing the PVA sponge is very useful for analyzing granulation tissue formation, collagen deposition, wound fluid composition, and the effects of substances on the healing process1,2,5. In addition to its use in studying a wide array of attributes of wound healing, the PVA sponge has also been used in many other types of studies. It has been utilized to investigate tumor angiogenesis, drug delivery and stem cell survival and engraftment1,2,6,7. With its great alterability, prior extensive use, and reproducible results, the PVA sponge is an ideal model for many studies1,2.
Here, we will describe the preparation, implantation and retrieval of PVA sponge disks (Figure 1) in a mouse model of wound healing.

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

聚乙烯醇海绵模型植入

Wound healing is a complicated, multistep process involving many cell types, growth factors and compounds1-3. Because of this complexity, wound healing ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

矫形肝移植大鼠模型中动脉重联后并发症减少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

采取下一步:神经整形正交后肢移植模型，以最大限度地提高大鼠的功能恢复

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...