Name: the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice
Uploaded: 2015-11-01T14:00:00.000+00:00
Duration: 12 min 4 s
Description: Watch this Scientific Journal Video about The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice at JoVE.com

This work was supported in part by the Lupus Research Institute, NCI grant CA138617, NIDDK grant DK090978, Charlotte Schmidlapp Award (to E.M.J.), and the Albert J. Ryan Fellowship (to J.K.). We are grateful for the support and instrumentation provided by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children&#8217;s Hospital Medical Center, supported in part by NIH AR-47363, NIH DK78392 and NIH DK90971.

根据该协会的评估和实验动物国际认证与我们的机构动物护理和使用委员会（IACUC）制定准则无特定病原体条件下进行动物实验。 注:表达同类标记纳入小鼠如CD45.1在任供体或受体动物如果可能的话，因为这允许对供体接枝效率和供体的CD4 T细胞群体的特定扩展的监测。如果考虑使用否则转基因小鼠作为供体或收件人，确保应变正确回交至B6的背景，或传送细胞可能会被拒绝，这将在代表性的结果和讨论部分加以处理更详细。 注:以下步骤详细说明卷收获4捐助BM12小鼠，这应该产生足够的细胞注入小鼠12-16。六到十二周龄BM12小鼠产生大约25％的CD4 T细胞大约100-140万美元的淋巴细胞。如果在启动不同数量的小鼠，或不同的小鼠品系，规模量向上或向下相应。 C57BL / 6小鼠一般给类似的产量与接近只有20％的CD4 T细胞。 注:执行所有的步骤在室温，用RT媒体，以避免冷热冲击，这会损害长期的淋巴细胞活力。执行组织收获，并使用无菌技术在组织培养罩组织处理步骤。在这个协议中的所有媒体是IMDM有10％热灭活FBS，除非另外指出，否则，将被简单地称为"完全培养基"。 1.新方法BM12基因分型小鼠注:这里提供一个简单的限制性酶切为基础的协议进行基因分型，为简单和廉价的替代测序，这是目前唯一公布的基因分型方法对这些小鼠。 <ol><li>隔离从小鼠尾巴基因组DNA。请参照详细的协议鼠标尾部剪裁以前的朱庇特的文章，并从尾部产生的cDNA消化13。 </li><li>执行逆转录聚合链式反应（PCR）14以扩增从MHC-II IA b和 IA一个共同的474 bp的DNA片段BM12使用表 1中所列的引物和热循环条件。 </li><li>执行的限制性消化上〜7微升PCR产物利用酶PsuI或其裂酶（BstX2I，BstYI，MflI，或XhoII），其切割野生型IA b，一个 ，但不是IA BM12突变体。遵循消化由生产，这将指定的水的混合物，缓冲液和酶，和5分钟的温育提供协议。至在37℃下15几个小时。 </li><li>加载消化产物并用溴化乙锭14上运行1％聚丙烯酰胺凝胶30-45分钟。在150V-虽然最佳电压和时间设置将根据所用的PCR凝胶装置而有所不同。可视化DNA带用UV照射。代表性的结果示于图1。 </li></ol><img alt="图1" src="/files/ftp_upload/53319/53319fig1.jpg" /> 图1.代表BM12基因分型结果。 为了鉴定小鼠纯合子IA BM12 / BM12，尾DNA中筛选BM12通过PCR /限制性消化的基因分型（步骤1）。纯合野生型（IA B / B）的DNA产生两个频段在227和247基点（可视化为一根粗带位于〜250基点）;纯合子BM12（IA BM12 / BM12）DNA产量为474 bp的一条带;和杂合子（IA BM12 / B）的DNA产生了两个带在〜250和474基点。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.收获捐助细胞<ol><li>使用实验动物管理和使用委员会（IACUC）牺牲供体小鼠-approved小学和中学安乐死方法。例如，安乐死的小鼠窒息用CO 2颈椎脱位。 </li><li>使用无菌技术，收获脾和淋巴结（颈浅，下颌，上臂，腋下，肠系膜和腹股沟）到含有10ml完整的媒体在11-13 15ml试管。 注:请参阅详细的淋巴结清扫术协议16以前的报告- 18。这种模式也成功仅使用小鼠脾细胞转移，但是除了淋巴结显著降低供体小鼠的所需数目。 </li><li>产生的单细胞悬液通过糖化使用从注射器柱塞通过70μm的细胞过滤器收集在步骤2.2淋巴组织。为了获得更好的收益，不超载过滤网使用〜6过滤器每4小鼠进行处理，并冲洗过滤器经常同时处理。从4只小鼠结合组织成两个50ml锥形管中，然后离心细胞5分钟，在400×g离心并倾析上清液。 </li><li>悬浮细胞从两个锥形管在50毫升完全培养基，并转移到新的锥形管中。保持这个管在室温。 注:脂肪附着在管的两侧，并且如果管不更换，最终细胞产率会受到影响。 </li></ol> 3.供体细胞计数注:为了保持整个样本的最高活力，红细胞（RBC）裂解不推荐。 <ol><li>混合的单细胞悬液从步骤2.4井，取出1 ml的，并将其传输到一个单独的50ml锥形。抛开其余的，不变的细胞（49毫升）在室温时计数。 </li><li>加入3毫升氯化铵基于红细胞裂解缓冲液，以1ml的供体细胞样品。轻轻混匀1分钟。摇动，然后填充到50毫升完整的媒体，一次离心5分钟。在400 XG，倒出上清液。 </li><li> （用血球例如，用台盼蓝）悬浮红细胞裂解的细胞在10毫升完整的媒体和计数。结果需乘49，这是剩下的被搁置非裂解样品中的细胞的总数量。放弃红细胞裂解的细胞。 </li></ol> 4.供体细胞标记和/或CD4 + T细胞纯化<ol><li>如果需要的话，净化的CD4 T细胞在此阶段，虽然这不是必需的。此外，如果需要的话，标签的细胞用CFSE，如在19或其它的细胞追踪染料，它的一个例子示出在代表性结果部分的图4。 注:对于CD4 T细胞的纯化，负磁选择被推荐的，因为它可以实现高纯度和如在20中所述叶细胞原封不动高生存能力。重要的是，无内毒素的缓冲器被使用;因此，代替牛血清白蛋白，使分离缓冲液的智慧H 2％FBS和EDTA的推荐的浓度。 </li></ol> 5.注射供体BM12细胞<ol><li>后，在400×g下计数供者淋巴细胞在步骤3（和纯化或标记为在步骤4中，如需要的话），离心机细胞5分钟。滗上清，重悬细胞于PBS中每毫升120万美元淋巴细胞（或30万美元纯化每毫升的CD4 T细胞）。转移细胞到无菌的5毫升的圆底管，或其它无菌管，可以轻松地容纳1ml注射器装配有27.5ģ×13毫米的针。 </li><li>在注射之前，预留供体细胞来自步骤5.1的小样品，在4℃下进行流动染色，以确定CD4 T细胞的供体样品中的百分比。作为使用最低抗体面板（表2）在步骤8中描述的染色这些样本。 注意:如果来自不同的小鼠品系细胞作为单独的供体，这是一个重要的考虑因素，并且如果CD4 T细胞的百分比显着变化，PURification可能需要。 </li><li>混合细胞轻轻地，彻底。这可以通过吹打细胞上下用1ml注射器没有连接的针来完成。混合后，绘制细胞在1ml注射器。去除任何气泡后附加针。保持针同时灌注注射器关有助于维持细胞活力。 </li><li>注入每只小鼠250微升（等于30万淋巴细胞，或者750万纯化的CD4 T每只小鼠细胞）腹膜内，如在21中所述。 注:在这里显示的实验，并在我们以前的工作11，每只小鼠的BM12注入捐赠者以30万总淋巴细胞，而不是传统上使用100万总脾细胞。在我们的实验室未公布的数据，没有什么区别，观察血清中抗dsDNA在以下每只小鼠30亿淋巴细胞注射14天。虽然这种显著减少所需的实验小鼠的数目，肾炎机智的发展H细胞的这个数字还没有评估。 </li></ol> 6.确定接枝效率注1:这部分将描述如何确定的供体细胞移植的程度在受体在第3天，以确定哪些可能已经收到了次优的注射任何小鼠（例如，在一个小鼠的移植物的中所见的&lt;10％所有其它小鼠来自相同组）。这些数据还可以帮助确定是否由转基因小鼠品系细胞在稍后的时间点拒绝（详见代表性的结果部分， 图6）。 注2:本节只有当供，受者使用CD45.1 BM12捐助者和CD45.2 C57BL / 6收件人时，从小鼠不同的同类背景，例如，或者当供体细胞进行标记细胞追踪染料。重要的是，第3天注射后，CD4 T细胞已经历最小膨胀（本身Ë代表性结果部分，图4），所以在接枝度观察到的差异是由于变异注射，不膨胀。 <ol><li>麻醉的小鼠用4％的异氟醚或其他IACUC批准的方法。测试后脚反射，以确保鼠标在进行抽血之前被适当麻醉。 </li><li>使用IACUC批准的方法，如后眼窝穿刺如在22收获100-200微升血液。收集血液到含有抗凝血剂，从个人0.5 ml微量离心管如在材料表中引用的等离子体收集管，其含有冻干二钾EDTA中。采血后，用无菌毛巾或纱布，以确保出血停止，然后将鼠标背部在其家笼在那里将继续，直到最后的组织收成（步骤7）施加温和的压力，鼠标眼。 注意:不要让老鼠无人值守，直到老鼠重新获得足够的意识保持腹卧，并不会返回老鼠给别人的公司，直到完全康复。 </li><li>带来血容量到500微升的21℃的PBS中，并转移到锥形底微量离心管中，然后慢慢下垫200微升21℃的高密度细胞分离溶液与200μl的移液管，小心以最小化两相之间的混合（见材料表推荐的解决方案）。离心细胞，在700×g离心20分钟在20-25℃下用离心机制动器设置为低。 </li><li>除去含有淋巴细胞1毫升吸管和转移到含有800微升冷的完整的媒体新的离心管的顶层。轻轻涡旋混合细胞。离心细胞，在700×g离心5分钟，在4℃和倾析上清液。 </li><li>悬浮细胞在200μl流式细胞仪如使用最小抗体面板（表2），以确定吨步骤8中描述的完整的媒体，转移到96孔U型底的板，和用于流污点他的CD4 T细胞的移植物的PBMC作为的百分比的相对丰度。 </li></ol> 7.最终组织收获注:在结果部分所述的实验中，收获注射的供体细胞后14天（或在某些情况下更短的时间），因为它们集中在TFH细胞和浆细胞的最初发展;然而，由于该模式是SLE的一种慢性GVHD模型中，疾病可在更晚的时间点进行监测。最佳的时间表将取决于所构成的每个实验所研究的问题。 <ol><li>在注射BM12供体细胞（步骤5.4）的后一个预定的时间点，使用IACUC批准的安乐死法牺牲小鼠。窒息用CO 2接着放血建议。颈脱位可以用作安乐死的辅助方法，但是这可能会降低血液抽取收率。 </li><li>润湿小鼠的腹部轻轻用含有70％喷雾瓶乙醇。做一个小的，肤浅的切口，手术剪约为1厘米以上的生殖器。退向胸骨腹部的皮肤，注意要保持腹膜筋膜完整。 注意:通常出现0.5-3毫升腹水在第14天，这，虽然还没有被很好地表征，可以测量和分析作为附加参数疾病患病的小鼠。 </li><li>适合5毫升注射器使用18G的针头。针插入有向向上朝向动物的头，并在15°角到筋膜平面的针腹部的右下象限。定位针尖端附近的盲肠，以帮助防止针越来越堵塞肠而抽吸腹水。 </li><li>小心地旋转在其一侧鼠标，然后慢慢吸取腹水入注射器。一旦腹水已被收回，取出注射器并在syri侧面记录分泌物量，根据容积标记NGE。 </li><li>放电腹水到一个5毫升的圆底管并存储在冰上供以后处理。 </li><li>通过从下腔静脉（IVC）基本上如在22拉伸收获血液。用平淡的镊子，轻轻移动肠子鼠标的左侧，揭露下腔静脉。插入一个27.5ģ针装配到1ml注射器入下腔静脉，并慢慢地吸取400-500微升血液。 </li><li>为了最大限度地减少溶血，缓缓注入血液进入0.5 ml离心血清或血浆收集管。对于抗核抗体ELISA法的后血清分析，血不断在冰上。 </li><li>解剖，将获得额外的相关组织（脾和，如果需要的话，淋巴结和肾脏，尤其是当收集在稍后的时间点和肾小球肾炎将得分）。轻轻放置肠子向后朝向右侧的动物，并拉动胰腺，这是脾脏的主要结缔组织除去脾脏。 </li><li>取出所有剩余的胰腺，然后称重脾脏的解剖后立即高精度平衡，总脾肿大是SLE的小鼠模型中常见的参数。将淋巴组织成单独的1.5毫升管填充用1ml完全培养基上冰。固定在10％中性缓冲福尔马林肾脏或卡扣冻结肾脏购买组织学在23。 </li><li> 2小时内收集的3分钟的离心血液。以10,000×g离心（4℃）。除去血清并储存在-80℃下的ANA通过ELISA以后分析。请参考详细ANA ELISA协议24,25以前的报告。在多个:10-20微升等份商店血清，以尽量减少冷冻/解冻循环，并允许更多数量的未来测定的。 </li><li>离心机腹水400×g离心5分钟。取出用1毫升吸管和分装上清分成几个0.5毫升管。冻结上清液在-80℃下购买抗核抗体（抗核抗体）或其它可溶性炎症介质的分析。 </li><li>悬浮细胞FRACT<html>离子在大约1 ml的完整的媒体和传送200μl到96孔U底板用于流染色（如在第8步）。 </li><li>每个捣烂脾通过70微米的细胞过滤到单独的管和冲洗完整的媒体。悬浮的脾细胞用1ml冷的RBC裂解缓冲液1分钟。并通过摇动轻轻混匀。把音量至10毫升冷的完整的媒体，离心5分钟400 XG，倒出上清液。 </li><li>在5毫升悬浮细胞沉淀完整的媒体和计数使用血球。调节音量，使200微升完整的媒体中含有1-3个细胞。开始染色使用扩展板（表2）来定量供体的CD4 T细胞和接收者B细胞分化和扩展流式细胞仪（步骤8）。 注:根据什么激光，光电倍增管（PMT），和过滤器组的组合是可用的，在T和B细胞分析面板分离成多个面板可能是必要的。 </li></ol>ve_title"&gt; 8。流程染色<ol><li>转印1-3百万脾细胞在200μl的完全培养基成单独的5毫升的圆底管，或到96孔U型底的板的单独的孔中。 注意:使用一个96孔板是染色多个样品，但照顾到板样品在每一个其它公为了防止交叉污染的有效方法。一个96孔板可以相应容纳24个样品。 </li><li>离心机板在500×g离心3分钟，然后轻拂上清液从板到合适的（生物危害）容器。 </li><li>悬浮含有可固定存活率染料在制造商推荐的浓度和纯化的抗CD16 /抗CD32抗体混合物在1微克/毫升的细胞沉淀用100微升流动缓冲液（1％FBS的PBS）中。孵育10分钟，在20-25℃下，再加入100微升冷的完整的媒体解渴活力染料。 注:从患病小鼠的脾细胞中通常含有比较高的数字死亡或濒临死亡的细胞;因此，夹杂物生存力染料的建议避免死亡细胞，导致更清洁，更可靠的数据的非特异性抗体标记。类似地，抗CD16 /抗CD32鸡尾酒包含阻断非特异性荧光标记的抗体通过Fc受体结合。 </li><li>离心机板在500×g离心3分钟，然后轻拂上清液从板进入生物危害容器。悬浮细胞在200μl流动缓冲液中。离心机板在500×g离心3分钟，然后轻拂上清液从板进入生物危害容器。 </li><li>悬浮细胞在50μl的流动缓冲器含有抗体混合物（ 表2）。孵育20分钟，在4℃下，再加入150微升流动缓冲液中。重复洗涤步骤8.4。 </li><li>悬浮细胞用在PBS中100μl的2％多聚甲醛。孵育30分钟，在4℃下，再加入100微升流缓冲区。离心机板在500×g离心3分钟，然后轻拂上清液从板进入生物危害容器。 </li><li> Resuspe次在200μl流动缓冲液，转移到流动管插入或标准流管，在黑暗中增加一个额外的100-200微升流动缓冲液，并储存于4℃，直至获取关于流式细胞仪。 </li><li>获得在流式细胞仪上配有相应的激光器和光电倍增管为选定的抗体的面板内染色的数天。除了记录前向散射的宽度和/或高度来转发散射区域，侧向散射区域，并利用荧光参数的区域。为可靠的结果，获得≥1,000供体细胞的每个WT样品中，或总淋巴细胞中，显示供体细胞，在收集的那么多的事件可能是行不通的最小扩展遗传修饰或以其它方式操纵的小鼠的等价数量。 注:流式细胞术分析中详细的代表性结果部（图3 - 6）中所述。 </li></ol>

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The authors have nothing to disclose.

的BM12诱导模型是研究SLE的细胞和分子过程相对容易且有效的方式。过继转移的CD4 T细胞直接针对自身抗原的慢性活化导致TFH，气相色谱B细胞和浆细胞的可通过流来测量仪，如这里所描述的积累。采用这种模式可以方便快捷地询问候选基因和新疗法的自身免疫生发中心的过程这类似于那些发生在SLE患者，最终支配自身抗体的病理积累的作用今后的研究。此外，这里所描述的流式细胞仪分析可用于研究涉及免疫球蛋白的发展，包括附加的小鼠模型中，但不限于自身免疫，感染和过敏。 如同人类疾病的所有动物模型中，这种模式也有其局限性。由于与疾病devel软件包速度OPS和其大小，不参与SLE的发展的所有基因都可能是必要的致病性在该模型中。此外，必须注意排除移植排斥反应时数据表明沙棘总黄酮对转基因收件人最小的扩张。同类系标志物应当用于确认供体细胞的存在在收获特别是由于受体细胞也可以分化成TFH（图3B），这可能会以其他方式掩盖不存在供体细胞。使用同类标记，我们观察到彻底消除供体细胞在第14天（ 图6），显然怀有排斥反应的抗原。我们已经成功地使用CD45.1 + BM12和CD45.2 + BM12小鼠为供体，并CD45.1 + BoyJ（B6.SJL- PTPRC 一个 PEPC B / BoyJ）和CD45.2 + C57BL / 6小鼠作为受体（图3，6，7，和未公布的数据）。然而，野生型同类CD90.1 +细胞从B6.PL- Thy1肾炎A / CYJ小鼠品系没有移植好，也不是一个CD90.1 +接收者（未公布数据）做CD90.2 + BM12细胞移植好了，这种现象显然是不独特这个模型26。 任一的C57BL / 6或BM12小鼠可作为供体或受体，如最初报告由Morris等人9然而，在我们的实验室，我们发现BM12细胞转移到B6小鼠产生的T细胞和B细胞的更一致的膨胀人口在14天此外，来自不同组供体和受体小鼠应该是性别和年龄相匹配的。虽然报道中的BM12模型的第一说明实验中发现的男性→男性和女性→女转账9任何疾病参数没有显著差异，男→女转让不建议，因为男性的抗原（HY）通过转染的CD4 T细胞表达可能诱发移植rejection女性收件人27。 当选择的抗体和荧光染料为同类系标志物，但是应当指出的是，供体细胞成为阳性为收件人同类系的标记在不同程度上，使得一个CD45.2 -门不会构成整个CD45.1 +移植物。该现象被清楚地示于CD45.2表达的CD45.1 +幼稚，CD45.1 +供体，和CD45.2 +接受者的CD4 T细胞（图7）的比较。值得注意的是，供体细胞似乎也上调自己同类系标志物的表达相比，幼稚对照（ 图7）。类似的结果也见于用CD45.2 + BM12转印到CD45.1 + BoyJ小鼠（数据未显示）。据推测，继多次收购从trogocytosis 28受体CD45结果由活化的CD4 T细胞受体与B细胞的相互作用。事实上，BM12模式升可能在研究trogocytosis的过程在体内证明的有用工具。 <img alt="图7" src="/files/ftp_upload/53319/53319fig7.jpg" /> 图7.供体细胞获得受体CD45同类指标。CD45.1 + BM12淋巴细胞转移到CD45.2 + C57BL / 6收件人。供体，受体和天真（CD45.1 +）CD4 T细胞转移后14天评估CD45.1和CD45.2表达。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 它已经显示，纯化的BM12的CD4 T细胞转移到C57BL / 7只小鼠是足以引发疾病29;然而，等效该病发展时整个淋巴细胞转移，和纯化不一定需要学习时Ť策疾病依赖LL-固有的基因表达。艾森伯格和他的同事，清楚地表明，在BM12模型中的抗体产生细胞是几乎完全收件人原点30的 。此外，对于受者的CD4 T细胞10的要求仅限于B细胞的其发展过程中的"培养"，可以是抵销通过加入外源性的IL-4 31，虽然我们的数据表明，受体T细胞可参与有些在生发中心的反应，因为他们中的一些做开发出沙棘总黄酮表型（ 图3B，右下图）。 必须为每个BM12实验中犯了一个重大的决定是什么时候收割的组织进行分析。当然决定取决于什么因素是很重要的目前的研究，这可能会发生变化。这里所描述的实验，需要长达14天，因为他们专注于TFH细胞和浆细胞的初步发展;然而，由于该模型是慢性SLE的GVHD模型，疾病可以更长时间监控。事实上，SLE的某些临床特征，包括肾小球肾炎，后来发展。蛋白尿已经检测早在2周后注射，但达到峰值水平在4-8周后注射10,31。此外，流式细胞仪分析在此描述了优化，用于实验持续2个星期。我们发现有相当数量的气相色谱B细胞和浆细胞在这个时间点的;虽然，给予额外时间，ANA分泌浆细胞的很大比例可驻留于骨髓32。此外，确定了这种流动染色板的浆细胞可能还包括浆母，以这两种细胞类型进行区分，另外的抗体将是必需的。 TFH膨胀想必到达位于其上我们已经集中在14天窗后一些点的峰。然而，据我们所知，还没有研究报道BM12施主小区数目超过2周，或使用ð同类指标来跟踪过继转移细胞。

系统性红斑狼疮（SLE）是一种复杂的自身免疫性疾病用抗核抗体（ANA）的生产和肾小球肾炎prototypically特征。许多其他的后遗症，包括皮肤，心肺，肝和病变与疾病在一些个体相关联。在美国的流行率估计相差很大，从150,000-1,500,000 1,2，特别高的发病率在女性和少数族裔3。虽然SLE的病因已经很难辨别，它被认为是来自各种遗传和环境因素，在系统性自身免疫其中高潮的相互作用出现。 许多动物模型已被用于研究导致病情发作和进展的因素。 SLE的经典小鼠模型包括遗传上易感小鼠品系包括NZB x NZW的F1模型及其NZM衍生物中，MLR / lpr狼疮应变，并且BXSB /雅阿应变，和可诱导系统，例如作为pristane和慢性移植物抗宿主病（慢性GVHD）模型4。自身抗体产生的GVHD模型早期的报告中使用的各种小鼠品系或仓鼠株父到F1转移5 - 8;用于研究狼疮样疾病更常见的方法目前包括DBA / 2父→（C57BL / 6×DBA / 2）F 1，和这里描述的BM12传递模型。每个模型都有自己的警告，但他们一般都有一个共同的一套与人类疾病的临床特点相关的功能。在小鼠模型中最经常报告的参数包括脾肿大，淋巴结病，肾炎，ANA生产，并在细胞水平，T滤泡辅助细胞（TFH），生发中心（GC）B细胞和浆细胞的扩张。 H2 - - AB1 BM12 / KhEgJ（BM12）小鼠，应变我的诱导BM12模型由淋巴细胞从IA BM12 B6（C）过继转移实现dentical到C57BL / 6除了上MHC II类3个氨基酸的取代，插入IA b的C57BL / 6（B6）小鼠。捐助者的CD4 T细胞受援国的装甲运兵车Alloactivati​​on导致慢性GVHD的症状非常类似系统性红斑狼疮。具体地，这些包括扩展供体来源的TFH，扩大受者来源的GC B细胞和浆细胞，和生产抗核抗体，包括抗dsDNA，抗单链DNA，抗染色质，以及抗红细胞抗体9。随着时间的推移，受体小鼠发展与在肾小球，间质性的IgG沉积物，和肾脏10的血管区域相关性肾炎。我们最近证明，类似于人类疾病，也有对I型IFN在该模型11中的关键作用。值得注意的是，对于人的SLE的定义标准包括肾炎的SLE兼容的抗dsDNA的存在下发展的抗体12，这两者都是该小鼠模型的显着的特征。 有SE在自发模型BM12模型veral优势。经典模型发展SLE状标志自发依赖任一杂交小鼠品系，近交小鼠品系不上B6背景，或在B6背景大的基因位点，这使交叉敲除或以其它方式转基因小鼠困难且耗时。与BM12诱导模型中，遗传修饰的小鼠可用作任一供体或受体，从而允许更快速鉴定的细胞区室，其中特定基因可能是疾病重要的。此外，在BM12模型疾病发展的速度要快得多，只需要2个星期，直到抗核抗体的外观相比，数月为最自发模型。此外，在对比的是开发疾病在不同时间点的自发模型中，疾病发作和进展中的BM12→B6模型高度同步。这允许适当尺寸的组群，可以B中的代Ë用于介入或治疗策略在疾病发展的任何阶段。 下面是用于通过过继转移BM12淋巴细胞到C57BL / 6小鼠，或遗传变异体上的B6背景发起SLE状自身免疫的详细协议。此外，我们描述了流式细胞染色方案，用于枚举TFH，气相色谱B细胞和浆细胞，细胞类型与人类疾病相关的。重要的是，这些协议也可以被用于表征疾病在SLE最小鼠模型，并确定TFH，气相色谱B细胞和浆细胞中的其他疾病模型。

<table><tbody><tr><td>B6.SJL-Ptprca Pepcb/BoyJ</td><td>The Jackson Laboratory</td><td>001162</td><td>CD45.1+ BoyJ mouse strain</td></tr><tr><td>B6(C)-H2-Ab1bm12/KhEgJ</td><td>The Jackson Laboratory</td><td>001162</td><td>Bm12 mouse strain</td></tr><tr><td>FastDigest PsuI</td><td>Life Technologies</td><td>FD1554</td><td>Restriction digest enzyme for genotyping</td></tr><tr><td>1X RBC Lysis Buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>IMDM</td><td>GE Healthcare</td><td>SH30228.01</td><td></td></tr><tr><td>Plasma Separation Tube (PST)</td><td>BD</td><td>365974</td><td>Blood collection tube with Dipotassium EDTA</td></tr><tr><td>Serum Separation Tube (SST)</td><td>BD</td><td>365967</td><td>Blood collection tube with Clot activator / SST Gel</td></tr><tr><td>Ficoll</td><td>GE Healthcare</td><td>17-1440-02&amp;nbsp;</td><td>High density cell separation solution</td></tr><tr><td>Lympholyte-M</td><td>Cedarlane</td><td>CL5030</td><td>High density cell separation solution</td></tr><tr><td>GL-7-biotin</td><td>eBioscience</td><td>13-5902-82&amp;nbsp;</td><td></td></tr><tr><td>Streptavidin-BUV395</td><td>BD</td><td>564176</td><td></td></tr><tr><td>CD138-BV421</td><td>BioLegend</td><td>142508</td><td></td></tr><tr><td>CD4-BV510</td><td>BioLegend</td><td>100559</td><td></td></tr><tr><td>TCR&amp;beta;-BV605</td><td>BD</td><td>562840</td><td></td></tr><tr><td>CD45.1-BV711</td><td>BioLegend</td><td>110739</td><td></td></tr><tr><td>CD45.2-FITC</td><td>BioLegend</td><td>109806</td><td></td></tr><tr><td>PD-1-PE</td><td>BioLegend</td><td>135206</td><td></td></tr><tr><td>CD19-PerCP</td><td>BioLegend</td><td>115532</td><td></td></tr><tr><td>Fas-PE-Cy7</td><td>BD</td><td>557653</td><td></td></tr><tr><td>CXCR5-APC</td><td>BioLegend</td><td>145506</td><td></td></tr><tr><td>Fixable Viability Dye ef780</td><td>eBioscience</td><td>65-0865-18</td><td></td></tr><tr><td>CD4-BV421</td><td>BioLegend</td><td>100443</td><td></td></tr><tr><td>1.2 ml FACS tube inserts, racked</td><td>USA Scientific</td><td>1412-1400</td><td></td></tr><tr><td>BD Falcon&amp;trade; Round-Bottom Tubes</td><td>BD</td><td>352017</td><td></td></tr></tbody></table>

the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

患病小鼠发展脾肿大在短短的14天，表现出的脾脏2-3次健康小鼠的大小在大容量和细胞构成（图2）的条款。 脾依次选通光散射（FSC-A通过SSC-A），消除双峰（FSC-W或-H通过FSC-A），活细胞（活力染料染色的低），以及CD4 +TCRβ+（ 图中图3A）。供体细胞来自基于CD45.1和CD45.2（图3B，左下）受体细胞区别开来。供体细胞主要是采用一件T滤泡辅助（TFH）细胞表型，如特征为PD-1，CXCR5和Bcl-6和ICOS（图3B，C）的上调。受体CD4 + T细胞群的一部分也分化成TFH（ 图3B，右下图）。后的初始模断转移细胞的和/或迁移，供体来源的TFH的膨胀是对数，Reaching:10-20百万细胞在小鼠脾脏后注入（图3D）14天。 转移淋巴细胞CFSE标记表明，供者的CD4 T细胞活化后早期分化成TFH;基本上所有划分细胞天3,7观察到的，和14上调CXCR5和PD-1（图4）。增殖峰轮廓也表明，捐助者的CD4 + T细胞的比例相对较低接受alloactivati​​on和分裂。在第14天，大多数可检测供体细胞是那些分割超出区划可测量通过CFSE的最大数目。 来自供体的TFH的膨胀伴随着内源性的GC B细胞和浆细胞（图5A）的相应积累。相比，TFH在脾浆细胞积累延迟，没有表现出增加了幼稚动物上天3或7（图5B）。 Accordingly，抗核抗体没有前9天（数据未示出）容易检测到，但能够可靠地量化为14 11天。 通过使用同类系标志物，我们观察到排斥供体细胞在多种菌株。虽然这是小鼠时未充分回交到C57BL / 6背景上的共同的问题，我们也观察到排斥时BM12细胞转移到B6.PL- 大鼠Thy1 一个 / CYJ（CD90.1）小鼠是通常被认为是充分回交。因此，将小鼠常规地筛选了在天〜3通过流式细胞染色血样以评估初始的CD4 T细胞移植物的效率;我们知道，从CFSE增殖实验发现移植的细胞在这个早期时间点扩大很少。这些结果然后与从第14天收获得到的结果。在拒绝的例子情况下3000万CD45.1 + BM12淋巴细胞转移到Cardif<html> - / -已被回交C57BL / 6小鼠为12代的小鼠。在第5天，所有的小鼠显示等效接枝（循环的CD4 T细胞的2-3％），但在第14天，BM12供体细胞完全从遗传修饰的收件人（图6）消除。 <img alt="图2" src="/files/ftp_upload/53319/53319fig2.jpg" /> 图2.脾生长动力学注射BM12淋巴细胞后，脾切除术（左）后直接称重的高精确度的平衡。活细胞数目通过使用台盼蓝排除死细胞（右）血球计数测定。结果被描述为平均值±SEM，其中n = 9,4，3和6。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> JPG"/&gt; 对T滤泡辅助细胞扩张SLE。CD45.1 + BM12淋巴细胞的BM12模型图3.分析转移到C57BL / 6收件人和脾脏进行了分析7-14天后。 （A）的代表性的门控策略表示"淋巴细胞"（第一面板），"单电池"（第二面板），"活细胞"（第三面板），和"CD4 T细胞"（第四组）。 （二）供体细胞来自受体CD4 + T细胞的CD45.1和CD45.2染色尊贵和PD-1和CXCR5的表达分析。 （ 三）供体细胞通过TFH表型，由几种蛋白质通常与TFH相关的上调所示。 （D）的典型结果，显示供体来源的TFH的膨胀（定义为CD4 + CD45.1 + PD-1 + CXCR5 +活细胞）在天3,7和14后传送。结果描述一个什么恶意±SEM，其中n = 5，4，3，6，分别。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53319/53319fig4.jpg" /> 图4. PD-1和CXCR5中上调上分裂的细胞。BM12细胞注射之前到C57BL / 6的收件人用CFSE标记。代表性的流量图是在3，7所示为供者的CD4 T细胞，注射后14天。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53319/53319fig5.jpg" /> 图5.扩展脾浆细胞和GC B细胞。（A）的代表性的流图从幼稚小鼠的脾，或那些来自小鼠在14天后CD45.1 + BM12转移。浆细胞被定义为CD138 + CD19 低活细胞（上图）。 GC B细胞的CD19 + B细胞 ，其表达的GL-7与Fas（底部小图）的一个子集。 （B）的Quantitave数据表示脾浆细胞随着时间的积累。结果被描述为平均值±SEM，其中n = 5，4，3，6，分别。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig5large.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/53319/53319fig6.jpg" /> 图6. BM12移植物是由一些转基因受体小鼠拒绝。CD45.1 + BM12淋巴细胞转移至任一遗传修饰小鼠（上图）或C57BL / 6（底部小图）受体小鼠。小鼠放血5天后喷射并评估接枝效率。脾细胞从相同的小鼠小号进行了分析7-14天后。 <a href="https://www.jove.com/files/ftp_upload/53319/53319fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" fo:keep-together.within-page="1" style="top-margin: 20px"><tbody><tr><td> 底漆名称 </td><td colspan="2"> 序列（5&#39; - 3&#39;） </td><td> 退火温度 </td></tr><tr><td> Bm12F </td><td colspan="2"> CGTGGTCCCCGCTGTCCCCC </td><td rowspan="2"> * 65℃ </td></tr><tr><td> Bm12R </td><td colspan="2"> GGGCAGAGGGCAGAGGTGAG </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>分割</td><td>周期数</td><td>体温</td><td>持续时间</td></tr><tr><td> 1 </td><td> 1 </td><td> 95℃ </td><td> 2分钟。 </td></tr><tr><td rowspan="3"> 2 </td><td rowspan="3"> 40 </TD&gt; <td> 95℃ </td><td> 15秒。 </td></tr><tr><td> * 65 - 0.5°C /步</td><td> 15秒。 </td></tr><tr><td> 72℃ </td><td> 45秒。 </td></tr><tr><td> 3 </td><td> 1 </td><td> 72℃ </td><td> 10分钟。 </td></tr></tbody></table> 表1:引物和热循环条件为BM12基因分型的优化热循环条件将取决于所用的确切的试剂和仪器。这些条件都得到了优化为能在每一步改变退火温度的热循环仪，尽管该协议也可以变通使用静态退火温度。 <img alt="表2" src="/files/ftp_upload/53319/53319table2.jpg" /> 表2:抗体面板脾TFH，GC B和浆细胞识别通过流式细胞仪介绍的是我们已经成功地使用面板用于评估接枝效率在第3天（左）和T和B细胞的BM12细胞注射（中）后的分析在第14天。这些都得到了优化，可以与5激光器（355，405，488，561和640纳米）的LSRII。推荐滤波器设置每个荧光列（右），尽管这些可能不是每一台机器的最佳选择。取决于什么激光，光电倍增管，和过滤器组的组合是可用的，在T和B细胞分析面板分离成多个面板可能是必要的。

Watch this Scientific Journal Video about The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice at JoVE.com

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

the-bm12-inducible-model-systemic-lupus-erythematosus-sle-c57bl6

Nanyang Technological University

Research

JoVE Journal

Medicine

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

17.4K 次观看.  Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine. The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

视频: The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

科研

免疫与感染

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

CYP2D6动物模型：如何诱导小鼠自身免疫性肝炎

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

医学

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

神经科学

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.

分析易患狼疮的MRL/lpr小鼠的蛋白尿、白细胞肾浸润和蛋白质的肾脏沉积

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including ...

小鼠实验性自身免疫性脑脊髓炎的诱导和评价

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

作者聚焦：创建与雄性和雌性小鼠相关的多功能实验性自身免疫性脑脊髓炎模型 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

摘要

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此视频中的章节

SJL小鼠诱导实验性自身免疫性垂体炎

CYP2D6动物模型：如何诱导小鼠自身免疫性肝炎

髓鞘少突胶质细胞糖蛋白（MOG

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

分析易患狼疮的MRL/lpr小鼠的蛋白尿、白细胞肾浸润和蛋白质的肾脏沉积

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

摘要

探索更多视频

此视频中的章节

SJL小鼠诱导实验性自身免疫性垂体炎

CYP2D6动物模型：如何诱导小鼠自身免疫性肝炎

髓鞘少突胶质细胞糖蛋白（MOG

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

诱导BALB/c小鼠药物诱导的自身免疫性肝炎研究其致病机制

小鼠远端结肠造口术, 一种新的 C57BL/6 老鼠转移性结肠炎模型

分析易患狼疮的MRL/lpr小鼠的蛋白尿、白细胞肾浸润和蛋白质的肾脏沉积

两性多发性硬化症建模：MOG35-55 诱导的实验性自身免疫性脑脊髓炎

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎