Name: induction of drug-induced, autoimmune hepatitis in balb/c mice for the study of its pathogenic mechanisms
Uploaded: 2020-05-29T12:30:00.000+00:00
Duration: 11 min 36 s
Description: Watch this Scientific Journal Video about Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms at JoVE.com

Njoku博士要感谢医学博士Noel R. Rose博士的指导和富有洞察力的讨论，这些指导和讨论导致了该模型的制定。

所有程序均由动物护理和使用委员会批准。
1. 肝 S-100 胞质蛋白或 CYP2E1 表位的三氟乙酰化
注意:首先，制备三氟乙酰化S100（TFA-S100）和三氟乙酰基化CYP2E1表位（TFA-JHDN5）。因为免疫需要同源S100蛋白，并且需要BALB / c小鼠来产生免疫原。该制剂产生大量的免疫原;因此，预计每年执行此部分大约四次。将使用相同的方法来制造TFA-JHDN5。环氧乙烷2E1表位（JHDN5），GII/ FNN / 英国储备金/西九加/ 内部勘测/自由交易体系/三氟碳纳米管反洗钱/三通电阻，可以进行测序或购买。
<ol><li>分离肝脏的S100部分。
	<ol>
<li>在用40-60mg / kg氯胺酮与4-6mg / kg甲苯噻嗪混合的5-10 BALB / c小鼠镇静后，通过观察肌肉张力的减少和对疼痛刺激（例如脚趾捏（戒断反射）无反应）以及纠正反射丧失和睑反射丧失来确认适当的麻醉深度。</li>
		<li>使用显微外科剪刀，使用中线切口暴露腹腔内内容物，并在下腔静脉中做一个小切口以除去血液。</li>
		<li>将24号血管导管放入门静脉中，并在4°C的水浴中将40mL磷酸盐缓冲盐水（PBS）pH 7.4灌注肝脏10mL / min。 取出并称量混合的肝脏，并将其切成小块（10 - 15毫米）。 注意:腹膜内注射额外的氯胺酮/甲苯噻嗪（80mg / kg:8mg / kg），然后颈椎脱位并打开胸腔使肺部塌陷而诱导安乐死。这种方法为动物提供了最小的疼痛和不适。这种方法符合美国兽医协会安乐死小组的建议。</li>
		<li>根据制造商的建议，加入4倍于蔗糖（250 mM）-TRIS（10mM）-EDTA（1mM）均质缓冲液（pH 7.4）的重量，并补充完全蛋白酶抑制剂鸡尾酒片（见 材料表）。在15 mL聚丙烯管中均质化，使用一般实验室组织均质机在冰上以中速搅拌直至光滑。在冰上均质化以防止组织在均质过程中变热。</li>
		<li>将肝脏以1500× g 离心匀浆10分钟，然后倒出上清液。将上清液以100，000×g离心1小时。快速冷冻上清液并储存在-80°C。 上清液为胞质S-100。</li>
	</ol></li>
	<li>S100 和 JHDN5 的三氟乙酰化反应 注意:S-100赖氨酸残基的ε氨基的三氟乙酰化将按照Satoh18的程序进行。除透析后期外，该实验的所有部分都在通风橱中进行。<ol>
<li>使用双新春酸测定（BCA测定）测定胞质S-100的总蛋白质浓度7。在 50 mL 锥形瓶中将 20 mg BALB/c 小鼠 S100 或 JHDN5 稀释至 10 mL，dH2O。用1N KOH将pH值调节至10。</li>
		<li>向溶液中加入4.7毫米的S-乙基三氟硫代乙酸酯（S-ETFA）。通过以液滴方式施用KOH约1小时，将pH值保持在9.9 – 10.0之间，并给予1N KOH。记录每个反应的KOH总体积。</li>
		<li>将溶液转移到单独的透析盒中（不要过量填充）。将暗盒透析72小时，对4升dH2O，每天更换三次。透析后，记录TFA-S100或TFA-JHDN5的最终体积，然后等分试样到标记的试管中。快速冷冻并储存在–80°C。</li>
		<li>通过将S100或JHDN5的初始量（以毫克为单位）除以透析后的最终体积（mL）来确定估计浓度。为了确定天然蛋白19的修饰百分比，在单独的子弹管中用dH2O将每种天然和TFA改变的蛋白质的1.0mg（如果最终浓度大于1.0mg）稀释至1.0mL，并使用1.0 mL dH2O制备空白。如果TFA改变的蛋白质的浓度小于1.0毫克，不要稀释<ol>
<li>为了分离96孔板的孔，加入50μL空白，天然和改变的蛋白质。向每个孔中加入50μL4%NaHCO3 ，然后加入50μL0.1%2，4，6-三硝基苯磺酸。</li>
			<li>将板在40°C孵育2小时。孵育后，向每个孔中加入50μL10%SDS，然后加入25μL1N HCl。</li>
			<li>在334nm的OD下读取，然后记录200 – 600nm范围内每种化合物的吸光度，以确认334nm处吸光度的特征下降。使用以下公式计算TFA对赖氨酸残基的修饰百分比: <img alt="Equation 1" src="/files/ftp_upload/59174/59174eq1.jpg">
</li>
		</ol>
</li>
	</ol></li>
</ol>2. 小鼠免疫诱发肝炎
注意:DIH在BALB / c小鼠中通过用肝细胞质蛋白进行免疫来建模，这些蛋白已被三氟乙酰氯（TFA），模型药物代谢物TFA-S1006 或CYP2E1的表位共价改变TFA9，TFA-JHDN5诱导肝炎，自身反应性T细胞和CYP2E1自身抗体。小鼠在初始免疫后2周表现出脾活化阶段，在3周时表现出肝期，其特征在于粒细胞炎症。在该模型中，雌性BALB / c小鼠比雄性更容易感染肝炎。
<ol><li>在第0天，用200μgTFA-S100或100μgTFA-JHDN5乳化在等体积的完全弗氏佐剂（CFA）中，在颈部底部皮下免疫6-8周龄的BALB / c小鼠。在第0天，用50ng百日咳毒素对小鼠进行免疫，在其中一条后腿肌内注射。在第7天，用200μgTFA-S100或100μgTFA-JHDN5乳化在等体积的CFA中，皮下在尾巴底部对小鼠进行免疫，以确保每只小鼠接受两次相同的免疫原注射。 注意:在免疫接种后的前6小时内每小时监测一次小鼠，然后每天至少监测两次，持续一周。如果小鼠表现出疼痛或痛苦的迹象，例如驼背的姿势或褶皱的皮毛，应根据当地的ACUC施用镇痛药。如果注意到明显的疼痛或痛苦，应由兽医评估小鼠以确定是否需要安乐死。</li>
	<li>使用流式细胞术测定CD4 + T细胞对整个自身蛋白，自身蛋白表位或TFA半抗原的免疫反应
	<ol>
<li>通过腹膜注射用40-60mg / kg氯胺酮与4-6mg / kg甲苯噻嗪混合的小鼠镇静后，按照步骤1.1.1所述确认适当的麻醉深度，然后使用显微外科剪刀暴露腹腔后识别脾脏。切开椎弓根处的脾脏，放入装有PBS / 2%胎牛血清（FCS）的培养皿中。 注意:腹膜内注射额外的氯胺酮/甲苯噻嗪（80mg / kg:8mg / kg），然后颈椎脱位并打开胸腔使肺塌陷，从而在小鼠中诱导安乐死。这种方法为动物提供了最小的疼痛和不适。这种方法符合美国兽医协会安乐死小组的建议。</li>
		<li>使用磨砂载玻片释放细胞并转移到50 mL锥形聚丙烯管中。用PBS/2%FCS洗涤，将体积提高到50 mL，并使用台式冷冻离心机以335 x g 离心。倒出上清液并重复此步骤。</li>
		<li>使用1 mL ACK裂解缓冲液除去红细胞1分钟，并用PBS / 2%FCS使体积高达50 mL。以335× g 离心并倒出上清液。</li>
		<li>对细胞进行计数。按照制造商的说明，在黑暗中用CFSE标记细胞30分钟。将单细胞悬浮液悬浮到PBS / 2%FCS中每孔3x10 6个细胞/ mL的6 孔板中。</li>
		<li>用CYP2E1，JHDN5或TFA-OVA（10μg/ mL）在37°C下在5%CO 2，95%空气（加湿）中刺激标记的细胞72小时。孵育后，用CD4-APC（1:100）在冰上染色细胞30分钟，并在3天内用流式细胞术分析。</li>
		<li>利用以下门控策略来鉴定CD4 + CFSE +细胞:CD4 + CFSE +细胞将从门控活细胞中鉴定出来，并显示为增殖细胞的直方图。</li>
	</ol></li>
	<li>从免疫小鼠中分离浸润的免疫细胞。 
	<ol>
<li>为了在第14天或第21天分离肝脏中浸润的免疫细胞，通过腹膜内注射用40-60mg / kg氯胺酮与4-6mg / kg木拉嗪混合麻醉小鼠，并确认步骤1.1.1中所述的麻醉深度。使用1.1.2中所述的用微手术剪刀制成的中线切口进行剖腹手术后，用25号针头囊状静脉穿刺，然后切除肾静脉下方的下腔静脉。</li>
		<li>在37°C的水浴中以10 mL / min的流速与40 mL PBS一起灌注每个肝脏。 灌注后，使用微手术剪刀切开肝蒂处的肝脏，取出胆囊，然后在肺门处切开肝脏。</li>
		<li>使用20 mL无菌注射器杵和冷PBS在网状不锈钢筛上破坏肝脏。使用300目筛将产生的细胞悬浮液过滤到50 mL预灭菌的离心管中。使用冷PBS将每个悬浮液降至50mL，然后通过以370×g离心10分钟来洗涤悬浮液。</li>
		<li>弃去上清液，然后通过处理将每个沉淀池入新的50 mL管中（建议每只小鼠一个管，但是，如果样品合并，建议使用2-4个沉淀/管）。将混合的沉淀悬浮在45 mL珍珠棉35%（PBS）和100 IU / mL肝素中。</li>
		<li>在20°C下以500×g旋转每个管10分钟。 弃去上清液并将沉淀悬浮在5mL PBS中，然后将1mLACK裂解缓冲液加入每个沉淀中，在冰上放置10分钟。</li>
		<li>将每个管与PBS一起带到50 mL，并以370×g离心10分钟。弃去上清液，然后用PBS / 2%FCS以370×g离心10分钟洗涤细胞。对细胞进行计数。</li>
		<li>
使用流式细胞术分析细胞类型 注意:以下是如何检测诱导的Foxp3 + 特雷格的示例。<ol>
<li>用FcR阻断试剂孵育1x106 个细胞，并用1:100稀释的CD4-FITC，CD25-PE和CD45-PerCP染色30分钟。接下来，用FoxP3-APC在细胞内染色细胞。</li>
			<li>用250μL固定缓冲液固定细胞（参见 材料表），并储存在4°C，直到3天内通过流式细胞术分析。</li>
			<li>建议使用流式细胞术检测来自肝脏、脾脏或淋巴结的单细胞悬浮液中诱导的 Foxp3+Tregs 的门控策略:使用活/死固定水死细胞染色试剂盒鉴定活细胞。接下来，对 CD45+（PerCP，克隆 RA3-6B2）的肝细胞进行门控，并对来自 CD45+ 门的 CD4+ 细胞（FITC、克隆 GK1.5）进行门控。从CD4 +细胞中，鉴定CD25 +（PE，克隆7D4）和FoxP3 +（APC，克隆3G3）细胞的百分比。</li>
		</ol>
</li>
	</ol></li>
	<li>肝组织对肝炎的组织学分析
	<ol>
<li>在第21天，将肝脏切片（5μm厚）固定在10%中性缓冲福尔马林中，并用苏木精&EOSIN染色。</li>
		<li>在平均 2 次查看中，首先在低功率 （40X） 下确定组织学评分，并在 64X 时确认。组织切片评分如下:0级=无炎症或坏死;1级=轻微小叶炎症，无坏死;2级=小叶炎症，涉及<50%的切片;3级=小叶炎症，累及≥50%的切片;和4级=炎症伴坏死。</li>
	</ol></li>
	<li>测定脾脏和肝脏中的组织细胞因子水平。 
	<ol>
<li>在第14天或第21天，将每只小鼠的肝脏或脾脏样品（1g）在1 mL RPMI / 2%FCS中均质化，直到在培养基设置上使用一般实验室均质器使其光滑。将样品放在冰上冷藏。</li>
		<li>使用冷冻台式离心机在4°C下以1455×g离心匀浆15分钟。快速冷冻上清液并储存在-80°C直至准备使用。细胞因子和趋化因子水平可以使用商用ELISA试剂盒根据试剂盒说明进行测量。通过将细胞因子的水平（以mL或μL为单位）转换为组织的pg / g来标准化细胞因子的水平。</li>
	</ol></li>
	<li>检测血清抗体，以检测细胞介苗2E1，CYP2E1表位JHDN5和TFA药物代谢物。
	<ol>
<li>在第14天或第21天，用40-60mg / kg氯胺酮与4-6mg / kg甲苯噻嗪混合镇静小鼠。通过观察肌肉张力的降低和对疼痛刺激（如脚趾捏（戒断反射））无反应，以及矫正反射的丧失和睑下反射的丧失，确认麻醉的深度。利用连接到结核菌素注射器上的25G针头，通过将针头缓慢推进到肋骨下方的胸腔并立即向剑突外侧来接近心脏。使用心内穿刺收集血液。</li>
		<li>一旦血液被收集，让它在室温下凝结。在室温下以295× g 离心血液样品20分钟。小心地除去血清，等分血清并在-20°C下快速冷冻。</li>
		<li>将100μLCYP2E1，JHDN5或TFA-卵清蛋白（OVA）测试抗原（PBS中为5μg/ mL）施用于96孔板中，在4°C下至少18小时过夜。第二天，用洗涤缓冲液（PBS / 2%FCS）洗涤板，2个周期（每个循环4次洗涤）。</li>
		<li>在PBS / 2%FCS中一式三份在平板上施用100μL小鼠血清（1:100），并在室温下孵育2小时。2小时后，按照步骤2.6.2中所述，用洗涤缓冲液洗涤板。</li>
		<li>加入100μL碱性磷酸酶（AKP）-山羊抗小鼠IgG，AKP-大鼠抗小鼠IgG1或AKP-大鼠抗小鼠IgG2a二抗（1:1000）2小时，然后用洗涤缓冲液洗涤1个周期和1个周期的PBS洗涤步骤。使用AKP底物试剂盒检测抗体，并用分光光度计每15分钟在OD 405nm处测量一次。TFA通常在15分钟内完全发展，而CYP2E1和CYP2E1表位可以在30至60分钟内7发展。</li>
	</ol></li>
	<li>研究JHDN5 IgG诱导的体外氧化应激的发展。
	<ol>
<li>使用12孔板，在1000μL补充谷氨酰胺和一般补充剂（参见材料表）的1000μL威廉姆斯E培养基中，在37°C，5%CO2，95%湿度下，每孔孵育106个终末分化的肝细胞，以达到7天的推荐值，以最大限度地提高CYP2E1活性。</li>
		<li>加入JHDN5 IgG（1:40）或小鼠IgG（1:1000）以分离孔并在37°C，5%CO 2，95%湿度下孵育2小时。杂交瘤血清非常稀释。向所有孔中加入深红色荧光抗体检测器（参见 材料表）30分钟。</li>
		<li>在黑暗中用1 mL PBS洗涤孔3x。将细胞固定在3.7%甲醛中10分钟。在24小时内通过共聚焦显微镜检查。</li>
	</ol></li>
	<li>JHDN5 IgG与细胞内细胞器（如线粒体）的体外共定位研究。 
	<ol>
<li>为了证明JHDN5 IgG与线粒体的共定位，在纤连蛋白覆盖的覆盖物上稀疏培养（〜30%汇合）终末分化的肝细胞在无染料的威廉姆斯培养基E中滑移7天，如步骤2.7中所述。</li>
		<li>确定正确的吸收波长后，加入绿色荧光，488nm共轭小鼠IgG或JHDN-5（1:100）和红色荧光，594共轭水母跟踪器红色（1:100）2小时（37°C），5%CO2，95%湿度。</li>
		<li>使用带有DAPI的抗褪色试剂安装标记的纤连蛋白覆盖的盖玻片，并通过共聚焦显微镜检查。</li>
	</ol></li>
</ol>3. 一般协议说明
<ol><li>当临床使用配方中没有化合物时，请使用非药物级工具。但是，从此方法中确定的可靠商业供应商处获取这些工具中的每一个。始终使用符合美国化学学会分析试剂委员会定义的至少试剂级规格的化学品。对于我们的方法，请尽可能使用分析级试剂。</li>
	<li>遵循严格的无菌技术来配制TFA改变的蛋白质，以防止可能对动物福利或数据解释产生不利影响的污染。</li>
	<li>根据可用的技术信息，在制剂保持效力的持续时间内储存和使用非医药级制剂。将CFA储存在室温下，CYP2E1及其表位在-20或-80°C。 将TFA改变的蛋白质储存在-80°C，并在用CFA乳化之前使其达到4°C。将TFA改变的蛋白质储存在-80°C并储存在等分试样中，以防止重复冻融循环。</li>
</ol>

<ol>
	<li>Castiella, A., Zapata, E., Lucena, M. I., Andrade, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+autoimmune+liver+disease:+A+diagnostic+dilemma+of+an+increasingly+reported+disease.">Drug-induced autoimmune liver disease: A diagnostic dilemma of an increasingly reported disease.</a> World J. Hepatology. 6 (4), 160-168 (2014).</li><li>Bjornsson, E. S., Bergmann, O. M., Bjornsson, H. K., Kvaran, R. B., Olafsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence,+presentation,+and+outcomes+in+patients+with+drug-induced+liver+injury+in+the+general+population+of+Iceland.">Incidence, presentation, and outcomes in patients with drug-induced liver injury in the general population of Iceland.</a> Gastroenterology. 144 (7), 1419-1425 (2013).</li><li>Castiella, A., Lucena, M. 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K., Vakos, A., Cottagiri, M., Mantilla, J. J., Santhanam, L., Thomas, D. 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=Identification+of+a+Shared+Cytochrome+p4502E1+Epitope+Found+in+Anesthetic+Drug-Induced+and+Viral+Hepatitis.">Identification of a Shared Cytochrome p4502E1 Epitope Found in Anesthetic Drug-Induced and Viral Hepatitis.</a> mSphere. 3 (5), (2018).</li><li>Cho, J., Kim, L., Li, Z., Rose, N. R., Talor, M. V., Njoku, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+bias+in+experimental+immune-mediated,+drug-induced+liver+injury+in+BALB/c+mice:+suggested+roles+for+Tregs,+estrogen,+and+IL-6.">Sex bias in experimental immune-mediated, drug-induced liver injury in BALB/c mice: suggested roles for Tregs, estrogen, and IL-6.</a> PLoS. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+autoimmunity.">Sexual dimorphism in autoimmunity.</a> Journal of Clinical Investigation. 125 (6), 2187-2193 (2015).</li><li>Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., Gillette, J. R., Pohl, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+studies+on+the+mechanism+of+halothane-induced+hepatotoxicity:+immunohistochemical+evidence+of+trifluoroacetylated+hepatocytes.">Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes.</a> Journal of Pharmacology and Experimental Therapeutics. 233 (3), 857-862 (1985).</li><li>Habeeb, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+free+amino+groups+in+proteins+by+trinitrobenzenesulfonic+acid.">Determination of free amino groups in proteins by trinitrobenzenesulfonic acid.</a> Analytical Biochemistry. 14 (3), 328-336 (1966).</li><li>Christen, U., Burgin, M., Gut, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halothane+metabolism:+immunochemical+evidence+for+molecular+mimicry+of+trifluoroacetylated+liver+protein+adducts+by+constitutive+polypeptides.">Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.</a> Molecular Pharmacology. 40 (3), 390-400 (1991).</li><li>Christen, U., Quinn, J., Yeaman, S. J., Kenna, J. G., Clarke, J. B., Gandolfi, A. 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=Identification+of+the+dihydrolipoamide+acetyltransferase+subunit+of+the+human+pyruvate+dehydrogenase+complex+as+an+autoantigen+in+halothane+hepatitis.+Molecular+mimicry+of+trifluoroacetyl-lysine+by+lipoic+acid.">Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis. Molecular mimicry of trifluoroacetyl-lysine by lipoic acid.</a> European Journal of Biochemistry. 223 (3), 1035-1047 (1994).</li></ol>

作者没有什么可透露的。

该协议的优势在于其可重复性;因此，遵守建议的步骤至关重要。免疫原的配方对某些人来说可能是一个障碍;然而，我们已经使用文档中描述的表位重现了我们的模型，这消除了分离肝脏S100部分的需要。额外的表位或蛋白质很可能被改变，并在免疫接种后诱发肝炎;然而，我们描述了那些我们已经使用的蛋白质，并获得了可靠的结果。几种蛋白质已被证明在卤化麻醉剂暴露时是三氟乙酰化的。最有...

该方法的目的是描述在体内发展的药物诱导的自身免疫性肝炎的小鼠模型，并证明如何利用它来研究这种疾病的分子，免疫学和遗传基础。我们研究的长期目标是通过研究易感患者的DIH来揭示导致慢性肝脏炎症和损伤发展的机制。肝病和肝硬化是25至64岁成年人的第六大常见死因。特异质性 DILI，有时也称为药物诱发的自身免疫性肝炎 （DIH），是美国急性肝功能衰竭的第三大常见病因。DIH 是最常见的肝药物诱导的超敏化过程，见于约 9% 至 12% 的自身免疫性1 型肝炎患者。绝大多数 DIH 患者是女性 2，3，4。一种类型的DIH在给予卤化挥发性麻醉剂（例如异氟醚，七氟醚，去氟醚或氟烷）后在易感个体中发展。这些麻醉剂与其代谢的反应产物共价结合肝脏蛋白，从而产生能够引起过敏或自身免疫反应的新型自身抗原5。
对参与麻醉剂和任何形式的DIH发展的致病机制的研究以前一直受到缺乏密切模仿人类疾病诱导的动物模型的阻碍。我们开发了一种实验性的DIH小鼠模型，其特征类似于患者免疫介导的DILI。肝炎是通过用两种自身抗原之一的免疫诱导的，这两种自身抗原已被三氟乙酰氯（TFA）代谢物共价修饰，该代谢物是在细胞色素P450 2E1（CYP2E1）5的麻醉剂氧化代谢后形成的。一种自身抗原是肝细胞质 S100 肝脏部分，它是几种蛋白质6 的混合物，第二种自身抗原是 CYP2E1 的表位，由麻醉免疫介导的 DILI7 患者的血清识别。通过使用对实验性自身免疫性肝炎相对耐药的BALB / c小鼠，我们将我们的模型与C57Bl / 6J小鼠8中S100诱导的自身免疫性肝炎免疫模型区分开来。
由于其临床表现的多样性，DIH很难在患者中研究。转化实验模型提供了在体内和体外评估疾病发病机制的能力。目前，没有其他诱导DIH的替代方法可以在不使用动物的情况下充分检查体内或体外适应性或先天性免疫反应。此外，由于S-100或CYP2E1表位的三氟乙酰化似乎不会产生刺激性免疫原，并且我们通过用TFA改变的蛋白质免疫来诱导DIH，因此这些动物在免疫或其他程序之前不会接受乙醚，任何卤化麻醉剂，巴比妥酸盐或酒精，考虑到这些药物可能会改变我们正在研究的参数。即便如此，我们通过利用计算机模拟来确认我们发现的CYP2E1表位9 的结合偏好，并通过证明雌性BALB / c小鼠发展出更严重的DIH10来镜像涉及女性性别的人类DICH，从而减少了小鼠的使用量。
尽管 DIH 在患者中有不同的表现形式，并且在临床疾病研究中面临挑战，但反应性药物代谢物对天然蛋白的翻译后修饰是卤化麻醉剂11 之后的发病机制 DIH 中公认的关键机制。研究人员还承认CYP2E1是这一过程中的主要自原12，13。识别翻译后修饰的CYP2E1和其他肝脏蛋白的白细胞介素（IL）-4-上调CD4 + T细胞的作用是通过吸引嗜中性粒细胞，嗜酸性粒细胞和肥大细胞进入肝脏14来接受麻醉DIH的引发剂，并且该机制已在许多形式的DIH15，16中得到证实。诱导表达FoxP3的CD4 + CD25 + T细胞（Tregs）可降低DIH的严重程度，并且脾脏中这些细胞的相对缺乏会恶化DIH 10，7。因此，通过利用体内小鼠模型评估DIH在体内和体外的遗传，代谢和免疫机制，已经使理解DIH的大多数进展成为可能。
由于我们和其他研究人员已经发现了IL-4，嗜中性粒细胞和嗜酸性粒细胞在使用不同的小鼠模型启动DIH中的作用，我们相信这一观察结果支持了我们的论点，即无论使用DIH模型，肝炎和损伤都是由IL-4诱导的。我们方案的优势在于利用体内方法，包括雄性和雌性小鼠，以及重复组织学，CD4 + T细胞增殖测定和细胞因子。我们使用体外研究的优势在于，它们减少了所需的小鼠数量，同时提供了分离驱动DIH的细胞相互作用的方法。我们建议使用雄性和雌性小鼠，因为这降低了在解释结果时出现无意识偏倚的可能性，并增强了我们研究的转化潜力，因为DIH的发病率，患病率和严重程度在女性17中更高。我们建议从单一供应商处获得鼠标;然而，如果这是不可能的，从与转基因小鼠相同的供应商处获得窝交配对照或野生型小鼠。

<table><tbody><tr><td>0.1% 2,4,6-trinitrobenzene sulfonic acid (TNBS)</td><td>ThermoFisher</td><td>28997</td><td></td></tr><tr><td>AKP Substrate Kit</td><td>BioRad</td><td>172-1063</td><td></td></tr><tr><td>BALB/c mice</td><td>Jackson</td><td></td><td></td></tr><tr><td>CellTrace&amp;trade; CFSE Cell Proliferation Kit</td><td>ThermoFisher</td><td>C34554</td><td></td></tr><tr><td>CFA H37Ra</td><td>Becton Dickinson (Difco Bacto)</td><td>231131</td><td></td></tr><tr><td>FcR Blocking reagent</td><td>Milteyi</td><td>130-092-575</td><td></td></tr><tr><td>General supplement</td><td>ThermoFisher</td><td>HPRG770</td><td></td></tr><tr><td>HepaRG&amp;trade; cells cryopreserved</td><td>ThermoFisher</td><td>HPR GC10</td><td></td></tr><tr><td>Live/Dead Fixable Aqua Dead Cell stain kit&amp;nbsp;</td><td>ThermoFisher</td><td>L34965</td><td></td></tr><tr><td>NaHC03</td><td>Millipore Sigma</td><td>S5761</td><td></td></tr><tr><td>Percoll&amp;reg;</td><td>Millipore Sigma</td><td>P1644-1L</td><td></td></tr><tr><td>Pertussis Toxin</td><td>List Biologicals</td><td>180</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Various</td><td></td><td></td></tr><tr><td>Pierce&amp;trade; Protease Inhibitor Mini Tablets, EDTA Free</td><td>ThermoFisher</td><td>88666</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>JT Baker</td><td>3140-01</td><td></td></tr><tr><td>S-ethyltrifluorothioacetate (S-ETFA)</td><td>Millipore Sigma</td><td>177474</td><td></td></tr><tr><td>Slide-a-lyzer dialysis cassettes (10 K, 12 ml)</td><td>ThermoFisher</td><td>66810</td><td></td></tr><tr><td>UltraPure&amp;trade; SDS Solution, 10%</td><td>ThermoFisher</td><td>24730020</td><td></td></tr><tr><td>Williams Media E, no phenol red</td><td>ThermoFisher</td><td>A1217601</td><td></td></tr></tbody></table>

induction of drug-induced, autoimmune hepatitis in balb/c mice for the study of its pathogenic mechanisms

药物诱发的自身免疫性肝炎 （DIH） 是最常见的肝药物诱导的超敏化过程，见于约 9% 至 12% 的自身免疫性肝炎患者。绝大多数 DIH 患者是女性。由于缺乏模仿人类疾病的动物模型，这些性别差异的患病率的潜在机制尚不清楚。即便如此，人们普遍认为潜在的机制与人类白细胞抗原单倍型和性激素有关。相比之下，使用DIH小鼠模型，我们发现IL-4启动的CD4 + T细胞直接针对细胞色素P450 2E1的表位诱导嗜中性粒细胞，巨噬细胞和肥大细胞流入雌性BALB / c小鼠的肝脏。使用该模型，我们还表明IL-33诱导的FoxP3 +调节性T细胞在雌性和雄性小鼠中具有针对DIH的保护作用。该DIH模型是通过用CYP2E1表位对小鼠进行免疫诱导的，该表位已与与DIH相关的药物代谢物共价改变。这种表位被 DIH 患者识别。我们的方法诱导稳健且可重复的肝炎和自身抗体，可用于研究DIH的发病机制。虽然体内研究在处理不当时会在小鼠中引起过度的疼痛和痛苦，但体内模型的优点是能够评估大量小鼠的疾病发病机制。此外，可以使用侵入性程序研究改变的肝脏蛋白质的生物效应。在实验设计中添加体外研究允许在细胞水平上进行快速重复和机械分析。因此，我们将演示我们的模型方案以及如何利用它来研究DIH的体内和体外机制。

图1所示用于诱导DIH的免疫计划代表了颈部底部（第0天）和尾巴底部（第7天）所需的两次免疫接种。图2显示了使用CFSE响应CYP2E1，JHDN5，CYP2E1表位和麻醉剂的三氟乙酰基（TFA）代谢物在第14天获得的代表性增殖数据。图3显示了第14天获得的诱导CD4 + CD25 + FoxP3 + Tregs的门控策略和代表性流式细胞术分析。图4显示?...

Watch this Scientific Journal Video about Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms at JoVE.com

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

我们描述了BALB / c小鼠中的体内免疫，转化性肝炎模型，可用于研究药物诱导的自身免疫性肝炎的发病机制，包括该疾病中看到的性别差异。我们将描述该模型如何使用体内和体外实验技术演示可重复的分析。

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 ...

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Immunology and Infection

2.9K 次观看.  Johns Hopkins University. 我们描述了BALB / c小鼠中的体内免疫，转化性肝炎模型，可用于研究药物诱导的自身免疫性肝炎的发病机制，包括该疾病中看到的性别差异。我们将描述该模型如何使用体内和体外实验技术演示可重复的分析。

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

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

科研

免疫与感染

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 ...

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

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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) ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

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.

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 ...

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

小鼠自发性自身免疫性甲状腺炎模型的产生

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

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.

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

摘要

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此视频中的章节

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

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

诱导小鼠肠道炎症由效应CD4的过继转移

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

从小鼠在原代肝细胞培养物的炎性反应的诱导

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

小鼠自发性自身免疫性甲状腺炎模型的产生

通过将CBir1 TCR转基因CD4 ⁺ T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

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

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

摘要

探索更多视频

此视频中的章节

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

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

诱导小鼠肠道炎症由效应CD4的过继转移

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

从小鼠在原代肝细胞培养物的炎性反应的诱导

系统性红斑狼疮的BM12诱导模型（SLE）的C57BL / 6小鼠

小鼠自发性自身免疫性甲状腺炎模型的产生

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

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

通过将CBir1 TCR转基因CD4 ⁺ T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

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