The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1）制备细胞系： <ol><li>发展表达马了FcεRIα的RBL-2H3.1细胞系： <ol><li>使用基本的组织培养技术的单层细胞系，转用pEE6质粒父母RBL-2H3.1细胞，承载着马FcεRIα基因（GenBank：Y18204.1）28。加入2微克微升-1的质粒DNA，以0.8毫升细胞以1.2×10 7个细胞毫升-1的密度。使用0.4厘米electrocuvette然后立即在冰上孵育10分钟，电穿孔的细胞在250伏960μF。 </li><li>用含有0.4克遗传霉素G418硫酸盐媒体选择转化的细胞，然后通过用荧光的IgE抗体标记它们通过FACS剩余的活细胞进行排序。使用所得的RBL-2H3.1表达马FcεRIα细胞系为调查2,3。 </li></ol></li><li>预测试抗体致敏： <ol><l我&gt;收获的RBL-2H3.1表示从融合培养皿马FcεRIα细胞。洗，然后重悬浮细胞培养基中，以5×10 5细胞毫升-1的细胞密度。 </li><li>的兴趣增加了IgE与悬浮细胞，以1毫微克毫升-1的终浓度，然后板100微升的细胞在96孔板中在列1-6和孵育在37℃+ 5％CO 2 + 90％的相对湿度为16小时。经过培养时间，并进行释放试验前，检查井显微镜的良好融合和细胞粘附下。 </li></ol></li></ol> 2）释放试验： <ol><li>洗涤细胞： <ol><li>暖释放缓冲液（25mM PIPES，120毫摩尔氯化钠，5mM的氯化钾，0.04毫摩尔氯化镁和1mM氯化钙）在37℃下，以允许细胞轻柔洗涤。 </li><li>通过轻弹板以除去细胞培养基，并加入100μl的温暖洗涤细胞，3个7°C，释放缓冲区。重复两次。 </li></ol></li><li>抗原挑战： <ol><li>准备一个连续稀释的抗原0纳克毫升-1，0.1纳克毫升-1，1纳克毫升-1，10纳克毫升-1，100纳克毫升-1（NIP-HSA或DNP-HSA），1000纳克毫升-1，万纳克毫升-1释放缓冲区，并在37℃的温水。 </li><li>第二小区洗涤后，弃去培养基，并用100μl抗原溶液代替。确保在同一行中的孔中（A1-6例如）具有相同的抗原浓度加入到其中。 </li><li>加入0纳克毫升-1抗原设立阴性对照A行。添加渐增的抗原浓度下后跟曲拉通-X缓冲液（5％的Triton X-100）中排H细胞以裂解细胞中的行（BG）被用作阳性对照。孵育在37℃下20分钟以使细胞释放其调停。 </li></ol></li><li>建立单井控制： <ol><li>培养后，转移50μl的细胞上清液，以在板的另一半（井A1-6至A7-12孔等）。丢弃剩余的50微升上清液，并用50μl的曲拉通-X缓冲替换，以允许在每个释放介质以及在列7-12的数量的测量作为细胞内的总介体在柱1-6的百分比。 </li></ol></li><li>酶底物： </li><li>添加50μl的β氨基己糖苷酶底物（50mM的4-硝基苯基N-乙酰基β-D-葡糖苷加入到柠檬酸盐缓冲液0.2M的柠檬酸和0.2M的醋酸钠制备在DMSO中稀释，以2毫米，pH值4.5）的到所有的孔中，以促进基片的转换，以4-硝基苯酚的β氨基己糖苷酶。孵育所述板在37℃下搅拌2小时。 </li></ol><img alt="图2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" widtH =“400”/&gt; <ol start="5"><li>终止反应： <ol><li>停止通过加入150微升的Tris缓冲液（1M的Tris-盐酸，pH为9），向每个孔中作为缓冲液的高pH值，反应停止反应并接通4-硝基苯酚的成黄色。 </li></ol></li><li>阅读和分析结果如下： <ol><li>使用平板分光光度计在405nm测定的黄颜色的吸光度读板。计算使用下面的公式释放β氨基己糖苷酶的百分比： </li></ol></li></ol><img alt="图2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li>应用此式向每个孔中，在这之后的平均值取为每一行。 A1和A7代表各孔中的96孔培养板的位置。积的β氨基己糖苷酶的释放（其对应于总的介质释放）的百分比的曲线图agaiNST抗原浓度2,3。 </li></ol>

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The authors declare that they have no competing financial interests in this paper.

在总结本研究的结果表明，当表达马FcεRIα的RBL-2H3.1细胞通过抗原致敏马IgE和挑战，他们得到的36.68％的峰值介质释放±介体内部的总量的4.88％细胞相比，所述的RBL-2H3.1亲本细胞不表达马FcεRIα。 因此该测定提供了用于调查研究马过敏反应体外的有用工具。其允许介由肥大细胞/嗜碱粒细胞谱系细胞释放的量的确定，从而可以预期到在马过敏推进研究，评估是否过敏致病?...

过敏已经知道了几千年。哮喘的治疗中被称为埃伯斯纸草（〜1550 BCE）的古埃及医书被描述和讨论草药治疗呢7。 今天过敏被分类为I型超敏性反应，其中，所述辅助性T细胞2型（TH 2）对免疫系统的臂牵引其生产免疫球蛋白E（IgE）的抗体响应于环境抗原称为变应原。这些是多种多样的物质，这些物质通常与细胞的相互作用在免疫系统，并刺激了合成和促炎性细胞因子的分泌，包括白细胞介素4和白细胞介素13 8,9作为别在香烟烟雾或柴油机排气颗粒颗粒增强IgE合成10。 在过敏表现上升工业化国家在过去50年来一直归因于效果的组合环境污染物和一种趋势，更加消毒环境中，它们组合转向由TH 2细胞因子占优势的轮廓的免疫应答，所建议的“卫生假说”11。 如上所述，人类不是由过敏折磨的唯一哺乳动物。特别是马和狗也可以开发经典过敏反应和研究12表明，在人类，马过敏是由于遗传和环境因素。其结果是，这些动物呈现良好的模型用于研究过敏的遗传和环境的原因，从致敏到疾病的进展，和可能的干预策略，一旦临床表现中已经设置之间的相互作用 1887年，Stömmer是第一个来描述人类和马哮喘13之间的相似性，组胺的马心血管系统的效果非常接近人类14。马也都是赛马业的基石，这是值得72美元十亿与115美元十亿年15投注额。 最现代的赛马都是小数量从1878年起，培育夫人安妮·布朗特阿拉伯马的后代。现代赛马通常近交系选择性能的能力。它们容易遗传性疾病，其中之一是其易安装过敏反应。它们还具有高1000倍的血清IgE水平甚至比最严重的过敏性人16。马过敏反应通常表现为昆虫叮咬过敏（IBH）17，18。IBH导致皮炎由于叮咬形成虫属中的库蠓 。马过敏性疾病的另一种形式是经常性的气道梗阻（RAO），这是表现在肺和呼吸道。它的特点是气喘和实验室或运算呼吸。饶通常发生在响应于模具孢子和高变应原特异性IgE水平已经记录在马从饶在一项研究中19痛苦虽然另一个调查还没有证实这20。 研究马过敏围绕在监控中和马的IgE通过开发抗马的IgE单克隆抗体（mAbs）21，22的企图。此外，研究了23讨论了生产的马的高亲和力Fc受体的α的胞外结构域的链（FcεRIα）受体，以试图检测和定量的马血清IgE。一项相关研究Ledin 24讨论了一种新方法，旨在中和血清IgE通过使用自体/非自吸免疫的免疫系统。所有这些研究中，但是，缺乏有效的测定法，以测试它们的协议的安全性和有效性。在这篇文章中，我们现在提出这样一个实验SYS统适用于相关的马系统，其中β氨基己糖苷酶的释放，作为细胞介脱粒的指标，进行了评估上表达马FcεRIα的RBL-2H3.1细胞诊断和治疗策略的研究。这个协议是基于以前的出版物25，4，5，2，3，描述用编码的高亲和力受体的IgE来自不同物种的IgE结合结构域的基因的RBL细胞的工程。该协议说明如何执行一个β氨基己糖苷酶释放测定，其结果表示为平均值±的一式三份实验的标准偏差。 释放法最早是由Siraganian开发和钩25来研究人类过敏。为首的鲁本Siraganian博士的实验室小组还开发出了RBL细胞系。这些RBL细胞开发了表达人FcεRIα和协议出版了4。最后一块的测定附带的PSV质粒在纸张其中所述的制造的IgE抗体的克隆及其小鼠基因的下游重链基因为靶向的半抗原4-羟基-3-一个的IgE可变区的发展由Neuberger的26硝基 - 苯乙（NP），将得到的嵌合抗体是全功能的。能力开发任何的IgE靶向相同​​的半抗原，同时也克隆及其受体导致使其成为一个有用的协议来测量嗜碱性粒细胞的脱颗粒测定的标准化的RBL细胞的表面上。 该法确实有优点和缺点。该测定法的优点是它的适应性，在任何哺乳动物系统中使用，我们的实验室已因而用它来测试在人，犬和马系统的脱粒，并且这是可以实现简单地通过合成的生物体的IgE和克隆受体到RBL细胞的表面上。 另一方面，该测定法的缺点是，RBL细胞对热，机械和PH值的变化非常敏感，使得它们得到脱粒水平的相同的测定范围内的变化。因此，这是强烈建议的分析总是重复一式三份，然后平均取自他们。此外，RBL细胞趋向于朝向非剥离性表型转变，如果它们被留在组织培养延长的时间（&gt; 10周）27，使得其维修很麻烦。他们还容易感染支原体的细菌，这是不是从一个光学显微镜可见的，不改变细胞的形态，但会极大地改变他们的脱颗粒的水平。因此，需要定期检查支原体。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

亲本的RBL-2H3.1细胞和那些转染了马FcεRIα受体基因进行了首次致敏的小鼠的IgE抗DNP-HSA和攻击用的DNP-HSA的抗原。小鼠IgE结合在两种细胞系中内源性大鼠受体并因此充当控制来测试这两种细胞系的生存力释放，释放介质（ 图1中的A）。这是一个重要的检查和常规自应进行后将延长（&gt; ​​10周），通过在细胞培养物，RBL-2H3.1细胞漂移朝向非分泌型27。亲本细胞支持的51.54％的峰值...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

的发展<em&gt;在体外</em&gt;模型研究的交互系统<em&gt;雅科仕</em&gt;<em&gt; caballus</em&gt;以其高亲和力受体了FcεRI的IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

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Immunology and Infection

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

12.3K 次观看.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.