用于炎症反应的新内皮调节因子的筛选测定
Summary
血管内皮严密控制白细胞的招募。不充分的白细胞外渗导致人类炎症性疾病。因此, 寻找新的调节元素内皮活化是必要的设计改进治疗炎症性疾病。在这里, 我们描述了一个全面的方法来表征新的内皮调节剂, 可以改变白细胞贩运在炎症。
Abstract
内皮层是必不可少的, 以维持体内的稳态, 通过控制许多不同的功能。调节炎症反应的内皮层是至关重要的有效打击有害的投入和援助, 恢复受损地区。当内皮细胞暴露在炎症环境中, 如 gram-negative 细菌膜的外成分, 脂多糖 (LPS), 它们表达可溶性炎细胞因子, 如 Ccl5, Cxcl1 和 Cxcl10, 并触发循环白细胞的活化。此外, 黏附分子 e-选择素、VCAM-1 和 ICAM-1 在内皮表面的表达能使活化白细胞与内皮层相互作用和黏附, 并最终向发炎的组织渗出。在这种情况下, 内皮功能必须受到严格的调节, 因为过量或有缺陷的活化在白细胞的招募可能导致炎症相关的疾病。由于许多这些疾病没有有效的治疗, 新的战略, 重点在血管层必须研究。我们建议全面的化验, 是有用的, 以寻找新的内皮调节剂, 改变白细胞功能。我们通过使用特定的表达靶点 (如细胞因子、趋化因子和黏附分子) 来分析内皮细胞活化作用, 包括: real-time 定量聚合酶链反应 (qPCR), 印迹, 流式细胞仪和粘附化验。这些方法可以确定炎症环境中的内皮功能, 对进行筛选化验以表征新的血管内皮炎症调节剂是非常有用的, 这对于设计治疗策略具有潜在的价值。
Introduction
炎症是对感染性药物的有益的生物学反应, 其主要目的是消除病原体, 修复受损组织。在某些情况下, 如慢性感染或自身免疫性疾病, 炎症无法解决。相反, 有一个异常的反应与持续的白细胞浸润, 导致长期免疫反应, 导致组织损伤, 纤维化, 功能丧失, 和整体, 残疾, 在某些情况下死亡的病人。这些人类疾病, 归类为炎症性疾病, 都涉及血管的控制白细胞外渗1,2。
内皮细胞通过控制白细胞的贩运在调节炎症反应中起着根本性的作用。当内皮层暴露在炎症介质, 如 LPS, 休眠内皮激活和表达炎细胞因子 (Cxcl10, Cxcl5, Cxcl1,等) 和黏附分子 (e-选择素, VCAM-1 和 ICAM-1), 有利于对感染部位进行循环白细胞的招募。由释放的细胞因子引导的白细胞, 然后调解滚动和互动的内皮层通过相应的胶粘剂对应: PSGL-1 的选择, α4β1整合素 VCAM-1, 和αLβ2整合素 ICAM-1。最后, 白细胞在血管中迁移到炎症的焦点3。
内皮细胞在调节炎症反应中所起的重要作用已经在基因修饰的小鼠身上得到证实, 它只在内皮细胞膜上表达 LPS 受体, 即 toll 样受体 4 (TLR4)。这些 endothelial-TLR4 动物能够对 LPS 介导的炎症反应和检测细菌接种后产生的感染, 从而达到与野生型小鼠一样的感染分辨率和生存率4,5。
对于内皮调节炎症反应通路, 已假设, 在某些阶段的白细胞-内皮相互作用的抑制将导致反式内皮细胞的迁移减少, 并有更好的预后炎症相关疾病。事实上, 一些针对内皮细胞活化和白细胞-内皮相互作用的策略被设计成阻碍免疫的外渗作为治疗炎症性疾病的方法6,7。
在本报告中, 我们描述了一个完整的组的体外技术, 以充分表征内皮活性响应炎症刺激 LPS 及其作用的白细胞活化和粘附到血管层。该手稿中使用的内皮细胞模型是小鼠肺内皮细胞系 (MLEC-04), 如 Hortelano et al.所描述的。8. 在文献中验证了 MLEC-04 细胞系是研究内皮细胞活化的适当系统9,10。根据研究兴趣, 这些方法可以很容易地推断出任何内皮或白细胞系统和炎症剖面。一旦确定了所选条件下的内皮参数, 系统就可以对新的药物进行实验, 以评价血管活化。在这种炎症的背景下, 用复利测定的内皮细胞可以与细胞的控制条件相比较, 任何结果的差异都可能使药物的预后与炎症的发展和进展有关。最后, 我们提出了一个相关的系统来表征新的药物靶向内皮细胞, 这可以影响设计的新型血管特异治疗炎症相关疾病。
Protocol
Representative Results
Discussion
这种内皮细胞的协议描述了一种逐步的技术, 建立了探索新的机制参与调节炎症反应的基础。这些方法是基于对 LPS 刺激的内皮活性的研究, 并评估在炎症反应过程中所涉及的白细胞招募的关键步骤, 特别是: 内皮细胞因子释放, 内皮黏附分子表达和白细胞粘附到血管层。一旦血管内皮细胞的参数建立, 系统可以寻找新的化合物参与调节内皮功能, 从而, 炎症进展。这些监管药物可能对医药市场有潜在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 Ministerio de Economía y Competitividad (MINECO) 和干杯卡洛斯 III (ISCIII) 的支持 (赠款编号 IERPY 1149/16 至 a.1;MPY 1410/09 到 s Hortelano);由 MINECO 通过基金 de 研究 en 干杯 () (授予数字 PI11.0036 和 PI14.0055 对 s Hortelano)。Herranz 由 ISCIII IERPY 1149/16 支持。
Materials
| Gelatin | Sigma | G9391 | |
| DMEM-F12 | Lonza | BE12-719F | |
| Fetal Bovine Serum | Sigma | A4503 | |
| Penicillin streptomycin | Lonza | DE17-602E | |
| Trypsine | Lonza | BE17-160E | |
| EDTA | Sigma | ED2SS | |
| LPS | Sigma | L2880 | |
| Trizol | Sigma | T9424 | RNA extraction buffer |
| Isopropanol | Sigma | 33539 | |
| Ethanol absoluto | Panreac | 1,310,861,612 | |
| Pure H2O | Qiagen | 1017979 | RNAse free |
| Agarose | Pronadisa | 8020 | |
| Stain for agarose gels | Invitrogen | s33102 | |
| SuperScript III First-Strand Synth | Invitrogen | 18080051 | Reagents for RT-PCR |
| Fast SYBR Green Master Mix | Applied Biosystems | 4385610 | Fluorescent stain for qPCR |
| MicroAmp Fast Optical 96-Well | Applied Biosystems | 4346906 | Plates for qPCR |
| U-bottom 96 well plates | Falcon | 353072 | |
| Cytometry tubes | Falcon | 352054 | |
| TX100 | Panreac | 212314 | Non-ionic surfactant |
| Tris-HCl | Panreac | 1,319,401,211 | |
| Sodium chloride | Merck | 1,064,041,000 | |
| Sodium pyrophosphate | Sigma | 221368 | |
| Sodium fluoride | Sigma | S7920 | |
| Sodium orthovanadate | sigma | 13721-39-6 | |
| Protease inhibitor cocktail | sigma | P8340 | |
| Pierce BCA Protein Assay Kit | Pierce | 23225 | Reagents for bicinchoninic acid assay |
| β-mercaptoethanol | merck | 805,740 | |
| PVDF Transfer Membrane, 0.45 µm | Thermo Scientific | 88518 | |
| Tween-20 | Panreac | 1,623,121,611 | Polysorbate 20 |
| PBS | Lonza | BE17-515Q | |
| ECL | Millipore | WBKLS0500 | |
| Fibronectin | Sigma | F1141 | |
| Laminin | Sigma | L2020 | |
| Collagen type I | Sigma | c8919 | |
| Acetic acid | Panreac | 1,310,081,611 | |
| Trypan blue | Sigma | T8154 | |
| Paraformaldehyde | Sigma | P6148 | |
| Methanol | Panreac | 1,310,911,612 | |
| Crystal violet | Sigma | HT90132 | |
| Sodium citrate | Sigma | C7254 | |
| Ethanol 96% | Panreac | 1,410,851,212 | |
| CFSE | Sigma | 21888 | |
| RPMI | Lonza | BE12-115F | |
| SDS | Bio-Rad | 161-0418 | |
| Infinite M200 | Tecan | M200 | Multi mode microplate reader |
| Gel Doc 2000 | Bio-Rad | 2000 | Gel documentation system |
| StepOnePlus | Applied Biosystems | StepOnePlus | qPCR system |
| MACSQuant Analyzer 10 | Miltenyi Biotec | Analyzer 10 | Cytometry equipment |
| ChemiDoc MP | Bio-Rad | MP | Chemiluminescence detection system |
| Name | Company | Catalog Number | Comments |
| Antibodies | |||
| PECAM-1 | BD Biosciences | 553370 | Use at 10 µg/ml |
| ICAM-2 | Biolegend | 1054602 | Use at 10 µg/ml |
| E-selectin | BD Biosciences | 553749 | Use at 10 µg/ml |
| VCAM-1 | BD Biosciences | 553330 | Use at 10 µg/ml |
| ICAM-1 | Becton Dickinson | 553250 | Use at 10 µg/ml |
| anti-rat IgG-FITC | Jackson Immuno Research | 112-095-006 | Use at 10 µg/ml |
| anti armenian hamster-FITC | Jackson Immuno Research | 127-095-160 | Use at 10 µg/ml |
| Rat IgG isotyope control | Invitrogen | 10700 | Use at 10 µg/ml |
| Armenian hamster IgG isotype control | Invitrogen | PA5-33220 | Use at 10 µg/ml |
| P-IκΒ-α | Cell Signaling | 2859 | Use at 10 µg/ml |
| β-Actin | Sigma | A5441 | Use at 10 µg/ml |
| P-ERK | Cell Signaling | 9101 | Use at 10 µg/ml |
| anti-mouse HRP | GE Healthcare | LNXA931/AE | Use at 1:10000 |
| anti-rabbit HRP | GE Healthcare | LNA934V/AG | Use at 1:10000 |
| anti-rat HRP | Santa Cruz | Sc-3823 | Use at 1:10000 |
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