在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝
Summary
某些鼠标菌株能够抵抗与髓鞘碱性蛋白诱导实验性自身免疫性脑脊髓炎（EAE）。这里描述的是一个简单的免疫协议，扭转反应迟钝和麻痹性疾病引起几个典型的EAE的耐鼠标污渍。
Abstract
实验性自身免疫性脑脊髓炎（EAE）是一种中枢神经系统（CNS）炎症性疾病，并已作为研究人类脱髓鞘疾病，多发性硬化症（MS）的动物模型。脑脊髓炎的特点是进入中枢神经系统的病理单核细胞浸润和麻痹性疾病的临床表现。类似于MS，EAE的是在某些小鼠品系遗传控制下也很容易受到疾病的诱导，而其他有抗药性。通常情况下，免疫C57BL / 6小鼠（H-B）与髓鞘碱性蛋白（MBP）的失败发展麻痹的迹象。此反应迟钝是肯定不会由于抗原加工缺陷或抗原的MBP介绍，这里描述的一个实验性协议已用于诱导C57BL / 6小鼠以及其他知名的抗小鼠品系严重脑脊髓炎。此外，脑炎C57BL / 6和BALB / c小鼠，以MBP的反应性T细胞克隆成功完全孤立和传播。
实验协议涉及使用蜂窝收养传输系统的MBP-底漆（200微克/鼠标）C57BL / 6捐助淋巴结细胞分离和培养五天与扩大池的MBP-特异性T细胞的抗原。在文化时期的结束，50万个活细胞通过尾静脉转移到天真同源收件人。通常这样对待的受体小鼠不发展脑脊髓炎，从而重申了他们的耐药现状，他们可以无限期地保持正常。十天后细胞移植受体小鼠挑战与完全弗氏佐剂（CFA）乳化四个网站在侧翼的MBP。严重的EAE的开始发展，在这些小鼠十至十四天后的挑战。结果表明，诱导疾病的抗原具体作为与不相干抗原挑战没有引起疾病的临床症状。值得注意的是，抗原剂量滴定挑战受体小鼠表明，它可能是为5微克/鼠标。此外，抗原挑战的时机动力学研究表明，早在5天抗原的挑战，如超过445天的抗原挑战，诱发疾病的挑战是有效的。这些数据有力地指向保持反应迟钝“长寿”的T细胞人口的参与。在这个系统中的调节性T细胞（Treg细胞）的参与，没有被定义。
Protocol
1。准备终审法院乳化的抗原在磷酸盐缓冲液（PBS）的2毫克/毫升的浓度，溶解豚鼠MBP（根据的方法Swanborg1的准备，并在冻干的形式储存在4°C）。如果合成的MBP肽（MBP的60-80 SHHAARTTHYGSLPQKSQR）2，准备2毫克/毫升的解决方案。达成鲁尔乐的玻璃注射器吸取适量抗原溶液（0.1毫升每鼠标进行免疫接种）。 画了一个完全弗氏佐剂H37Ra株（0.1wt％的）（终审法院，Difco公司实验?…
Discussion
在小鼠EAE的研究往往利用的neuroantigens，髓鞘碱性蛋白（MBP），蛋白脂蛋白（PLP）有或髓鞘少突胶质细胞蛋白（MOG）。早先的研究中大多采用的MBP。工党刺激SJL小鼠的强烈和一致的反应。最近，MOG是共同neuroantigen使用，因为很容易受到疾病与此抗原诱导B6小鼠。许多有针对性的小鼠品系的基因B6的遗传背景。有趣的是，B6小鼠容易与MOG诱导EAE，但抵抗疾病的MBP诱导。
许多以前的?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
从国立卫生研究院的资助（以R01 NS37253和N01，NS055167）和国家多发性硬化症协会（的RG 3288 – A6）在部分支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Guinea pig spinal cords | Rockland Immuno-chemicals, Inc. Gilbertsville, PA 19525 www.rockland-inc.com |
GP-T065 | frozen |
| Freund’s Adjuvant, complete H37Ra | Difco Laboratories Detroit, MI |
231131 | |
| Multifit interchange-able syringe | B-D www.bd.com/us |
512133 | |
| RPMI 1640 | Mediatech Inc. Manassas, VA www.cellgro.com |
10-040-CM | |
| OVA | Sigma-Aldrich www.sigmaaldrich.com |
A5503 | |
| Mice | Jackson Laboratory Bar Harbor, Maine |
B6 mice: 0000664 |
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Tags
Experimental Autoimmune EncephalomyelitisEAEMouse StrainsAdoptive TransferAntigenic ChallengeCentral Nervous SystemMononuclear CellsParalytic DiseaseMultiple SclerosisGenetic ControlC57BL/6 MiceMyelin Basic ProteinAntigen ProcessingAntigen PresentationEncephalitogenic T Cell ClonesCellular Adoptive Transfer System
Citazione di questo articolo
Shaw, M. K., Zhao, X., Tse, H. Y. Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge. J. Vis. Exp. (62), e3778, doi:10.3791/3778 (2012).