一个简单的生物测定血管内皮生长因子的评价
Summary
我们描述了检测，定量和监测血管内皮生长因子家族的配体的成员的活性的简单的基于细胞的生物测定法。该测定法使用在因子相关性细胞系中表达的嵌合受体的配体，以提供受体结合和交联的半定量或定量的评估。
Abstract
参与血管生物学受体酪氨酸激酶及其相互作用的配体的分析常常是具有挑战性的，由于相关的受体，范围广泛的相关配体和处理专门内皮细胞的原代培养物的难度家庭的组成型表达。这里，我们描述用于检测配体的血管内皮生长因子受体-2（VEGFR-2）中，促进血管生成和淋巴管生成的信号的键传感器生物测定。编码VEGFR-2的胞外（配体结合）区的融合与跨膜和促红细胞生成素受体（EpoR的）的细胞质区域的cDNA，在因子相关性细胞系的Ba / F3表示。该细胞系生长在白细胞介素-3（IL-3）和该因子的结果的撤出在细胞的死亡存在24小时之内。在VEGFR-2 / EpoR的受体融合的表达提供了一种替代机制，促进生存和势的在能够结合和交联的融合蛋白（ 即 ，一个可以交联的VEGFR-2的胞外区）的胞外部分的配位体的存在下稳定转染的Ba / F3细胞的LLY增殖。半定量的方法，其中配位体和细胞小体积在24小时允许快速结果，并且涉及活细胞数目的替代标志物的定量方法：在测定可以通过两种方式来执行。该测定是比较容易进行，是高度响应于已知VEGFR-2的配体，可容纳的VEGFR-2的信号，如单克隆抗体的受体或配体，和可溶性配体陷阱胞抑制剂。
Introduction
血管内皮生长因子（VEGF）家族的分泌蛋白的生长因子和它们的同源细胞表面受体是可溶性配体和膜包埋的受体，分别在穿过细胞膜转导信号该函数的一个重要和多样的基团。它们主要发挥作用的内皮细胞，而且在和上皮来源的细胞的那些免疫系统1,2。信令由配体激活的VEGF受体（VEGFRs）是在主要的病状，如年龄相关性黄斑变性和癌症关键的，和治疗啮合途径靶向它们是在常见的临床使用（例如，单克隆抗体贝伐单抗靶向的VEGF-A）的3,4。
一项所述的VEGF家族的复杂性是存在于自然界（可溶性配体VEGF-A，VEGF-B，VEGF-C，VEGF-D，由parapox病毒家族ORF和蛇毒VEGF，加上其他编码的VEGF蛋白质的多样性抑制VEGF-A）2亚型。
这些配体与受体酪氨酸激酶家族的三个成员，即VEGFR-1，VEGFR-2和VEGFR-3相互作用。这些受体可变地表达于不同的细胞类型，但该行的所有大小5的血液和淋巴管内皮细胞的表面上常常共表达。 VEGFR-2可以结合哺乳动物配体VEGF-A 6，VEGF-C 7和VEGF-D -8,9-以及口疮病毒VEGF 10和蛇毒VEGF的11。 VEGFR-2在驱动血管发生在胚胎发育（新血管从预先存在的血管的生长），伤口愈合，癌症和眼病主要作用。在这些情况下，配体例如VEGF-A，-C和-D结合并激活血液血管内皮细胞12-15受体。上淋巴管内皮细胞，VEGFR-2在淋巴管生成的作用，新的淋巴管16的形成。 VEGFR-2还可以促进扩张和主要动脉和淋巴管在健康组织和疾病17扩张。 VEGFR-2的完整的理解：配体的相互作用是因此对于抑制剂的开发用于治疗血管生成依赖性疾病18重要。而VEGF-A结合的对VEGFR-2的大部分同种型，是必需的VEGF-C和VEGF-D的蛋白水解裂解，以释放由表现出高亲和力结合VEGFR-2 19,20的VEGF-同源结构域的片段。
我们已经开发出一种生物测定来监测，其被设计来规避初级内皮细胞，这是技术上难以通过，购买昂贵的和培养的需要VEGFR-2的配体（需要专门介质）21和表达多种VEGFRs和相关的共受体22。旨在螺柱当与其他VEGF受体或共受体VEGFR-2的异源可引起不希望的复杂性ý二进制受体-配体相互作用，评估活性归属于特定的受体，或评估抑制试剂的效果。23。生物测定保留在细胞膜相关受体的流动性，并允许的配位体的结合能力并交联的VEGFR-2的细胞外区域的评价。
生物测定依赖于其中（在这种情况下，VEGFR-2）VEGF受体的胞外区融合至跨膜和促红细胞生成素受体（EpoR的），细胞因子受体家族的一个成员的胞内区域的创建的嵌合受体8,24。此融合蛋白然后在从属因子原B细胞系的Ba / F3表示，在其上的刺激与能结合和交联的受体的胞外结构域导致胞质效应区，其能够激活一个配位体通过JAK激酶（JAKS）转导存活信号，促进细胞的存活和/或增殖。与此相反，全长VEGFR-2在相同的细胞类型，并刺激配体表达，不促进细胞存活和增殖，表明VEGFR-2通路的近端信令效应不在此细胞类型可用。
我们已经使用该测定在各种情况下，探索新的VEGFR-2的配体10,19,20,24-29的结合。在与VEGFR-3-EpoR的-的Ba / F3测定结合，我们比较了VEGF-C的结合和交联的VEGFR-2和VEGFR-3 30的相对活性和VEGF-D的生长因子。该测定法已被用于表征中和单克隆抗体对VEGFR-2或VEGF-D，可溶性VEGFR-2捕集和拟肽靶向VEGF家族31的抑制活性。该试验也用于显示的VEGF的不同ORF病毒株结合交联的VEGFR-2在测试之前在初级内皮细胞的能力，并<s达> 10,26。该测定是对的VEGF的突变体的快速筛选，可以它们被引入到更费力内皮细胞分析25，或当用于净化生长评估协议因子27前被快速评估活动特别有用。
我们描述了检测容易进行，并且半定量版本允许监测的生长因子，抗体或其他实验的可溶性受体结构域的生产或纯化时有时需要快速测定。使用该测定的易用性使得它与从血液或淋巴管衍生自特定的组织或器官系统主内皮细胞进行进一步和更完全的研究，一个理想的补充。
Protocol
Representative Results
Discussion
此处所描述的测定法依赖于使用高存活率的细胞，这是依赖于生长因子。因此细胞需要仔细培养，以确保它们是因子依赖性的，并保留嵌合受体的表达。确保介质新鲜的，而不是存放过长的时间，并且WEHI-3D CM为高活性是重要的。细胞需要从含培养基的IL-3彻底洗涤进测定培养基，以确保无残留的IL-3将细胞暴露于配位体抢救时污染测定。作为配体可以来以多种不同的形式，护理需要用于测定制?…
Declarações
The authors have nothing to disclose.
Acknowledgements
SAS and MGA are supported by Project Grants, a Program Grant and Research Fellowships from the National Health and Medical Research Council of Australia (NHMRC), and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. MMH has support from a Peter MacCallum Foundation Grant.
Materials
| Trypan Blue | Sigma-Aldrich | T8154 | 0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer |
| G418 Sulphate (Geneticin) | Invivogen | ant-gn-5 | Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties. |
| 3H-Thymidine | PerkinElmer | NET-027 | This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation |
| Vialight Plus Kit | Lonza | LT07-221 | Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent |
| Prestoblue Cell Viability Reagent | Invitrogen | A13261 | Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell |
| Nunc Minitray with Nunclon Delta Surface (72 well) | Thermo Scientific | 136528 | Small microtitre plate |
| 96 well Tissue Culture Plate | Falcon, Corning Inc. | 353072 | |
| DMEM (1X) | Gibco | 11965-92 | |
| GlutaMAX (100X) | Gibco | 35050-061 | |
| Foetal Bovine Serum | Gibco | 10099-141 | |
| Cell Harvester | Tomtec Life Sciences | N/A | Tomtec Harvester, 96 Mach 3M Cell Harvester |
| Liquid Scintillation Counter | LKB Wallac | 1205 | LKB Wallac 1205 Betaplate Scintillation Counter |
| UniFilter-96 GF/B | Perkin Elmer | 6005177 | White 96-well Barex Microplate with GF/B filterof 1 µm poresize |
| Gentamicin | Gibco, Life Technologies | 15750-060 | |
| Penicillin/Streptomycin | Gibco, Life Technologies | 15140-122 | |
| 0.22um pore cellulose acetate centrifuge tube filter unit | Costar, Corning Inc. | 8160 | Centrifuge tube filters have a 0.22µm pore CA membrane-containing filter unit within a 500µl capacity polypropylene microcentrifuge tube. |
| Fluorescence Reader | BioTek | N/A | BioTek Synergy 4 Hybrid Microplate Reader |
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