β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析
Summary
本文介绍了两种方法, 可以单独使用, 也可以结合使用, 分析β-淀粉样蛋白对纤维蛋白凝块结构的影响。其中包括一种用于创建体外纤维蛋白凝块的协议, 其次是血栓浊度和扫描电子显微镜方法。
Abstract
本文介绍了用光谱法和扫描电镜 (sem) 法生成β-淀粉样蛋白 (aβ) 蛋白对凝块形成和结构的影响的体外纤维蛋白凝块的制备方法。aβ在阿尔茨海默氏症 (ad) 中形成神经毒性淀粉样聚集体, 已被证明与纤维蛋白原相互作用。这种 aβ-纤维蛋白原相互作用使纤维蛋白凝块结构异常, 对纤溶具有抵抗力。β诱导的纤维蛋白凝血异常也可能导致脑血管方面的 ad 病理, 如微梗塞, 炎症, 以及脑淀粉样血管病变 (caa)。鉴于神经血管缺陷在 ad 病理中的潜在关键作用, 开发能够抑制或减少 aca-纤维蛋白原相互作用的化合物具有很好的治疗价值。在体外的方法,其中纤维蛋白凝块的形成可以很容易和系统地评估是潜在的有用的工具, 开发治疗化合物。本文介绍了一种纤维蛋白凝块体外生成的优化方案, 以及 aβ和β-纤维蛋白原相互作用抑制剂的影响分析。凝块浊度检测快速、重现性高, 可用于同时检测多种条件, 从而可以筛选出大量的β纤维蛋白原抑制剂。可以进一步评估这种筛选产生的命中化合物是否能够使用 sem 改善光纤纤维蛋白凝块结构异常。使用 tdi-2760 (一种最近发现的抗逆转录酶原相互作用抑制剂) 在这里演示了这些优化方案的有效性。
Introduction
阿尔茨海默病 (ad) 是一种导致老年患者认知能力下降的神经退行性疾病, 主要源于β-淀粉样蛋白 (aβ) 的异常表达、聚集和清除障碍, 导致神经毒性1,2. 尽管 aβ集合体与 ad3之间存在良好的联系, 但疾病病理的确切机制尚不清楚4。越来越多的证据表明, 神经血管缺陷在 ad5的进展和严重程度中起着一定的作用, 因为 aβ与循环系统的组成部分直接相互作用。aβ与纤维蛋白原7,8有高亲和力的相互作用, 在 ad 患者和小鼠模型9,10,11中也定位到 aβ沉积。此外, β-纤维蛋白原相互作用诱导异常的纤维蛋白凝块形成和结构, 以及对纤维蛋白溶解的抵抗力9,12。治疗 ad 的一种治疗方法是通过抑制 aβ和纤维蛋白原13, 14 之间的相互作用来缓解循环缺陷。因此, 我们通过高通量筛选和药物化学方法 13, 14, 发现了几种抑制β纤维蛋白原相互作用的小化合物。为了测试光纤 b 系与原相互作用抑制剂的有效性, 我们优化了两种体外纤维蛋白凝块形成的分析方法: 凝块浊度分析和扫描电镜 (sem)14。
凝乳浊度测定是利用紫外可见光谱监测纤维蛋白凝块形成的一种直接快速的方法。随着纤维蛋白凝块的形成, 光线越来越分散, 溶液的浊度也在增加。相反, 当 aβ存在时, 纤维蛋白凝块的结构会改变, 混合物的浊度会降低 (图 1)。抑制化合物的效果可以评估的可能性, 以恢复凝块浊度从β诱导的异常。虽然浊度分析可以快速分析多种条件, 但它提供的关于凝块形状和结构的信息有限。sem, 其中固体物体的地形是由电子探针揭示, 允许分析的三维结构的凝块15,16, 17,18, 并评估如何存在的 aβ和/或抑制化合物改变结构9,14。光谱法和扫描电镜都是经典的实验室技术, 已被用于各种目的, 例如, 分光光度法用于监测淀粉样蛋白聚集19,20。同样, sem 也被用来分析由阿尔茨海默氏症、帕金森病和血栓栓塞中风 21、22、23 例患者的血浆形成的纤维蛋白凝块。这里介绍的协议是优化评估纤维蛋白凝块形成的重现性和快速的方式。
下面的协议提供了在有和没有 aβ的体外纤维蛋白凝块的制备说明。详细介绍了 aβ对纤维蛋白凝块形成和结构影响的分析方法。这两种方法测量抗β纤维蛋白原相互作用的有效性已证明使用 tdi-2760, 一种小型抑制化合物 14.这些方法, 无论是单独和共同, 允许快速和直接的分析体外纤维蛋白凝块的形成。
Protocol
Representative Results
Discussion
这里描述的方法为纤维蛋白凝块体外形成的评估提供了一种可重复和快速的方法。此外, 该系统的简单性使得对 aβ如何影响纤维蛋白凝块形成和结构的解释相对直接。在本实验室的前一份出版物中, 证明了这些检测方法可用于测试化合物抑制 ab-纤维蛋白原相互作用的能力 13,14。利用这两种方法, 分析了一系列合成化合物抑制β纤维蛋白原相互作用?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢纽约三机构治疗发现研究所 (tdi) 的川崎正森、麻岛一郎和迈克尔·福利合成了 acam 纤维蛋白原相互作用抑制剂及其宝贵的建议。作者还感谢斯特里克兰德实验室的成员进行了有益的讨论。这项工作得到了国家卫生研究院批准 ns104386, 阿尔茨海默氏药物发现基金会和罗伯逊治疗发展基金 h. a., nih 授予 ns50537, 三机构治疗发现研究所, 阿尔茨海默氏症药物发现基金会,rudin 家庭基金会和 john a. herrmann 为 s. s。
Materials
| Fibriogen, Plasminogen-Depleted, Plasma | EMD Millipore | 341578 | keep lid parafilm wrapped to avoid exposure to moisture |
| Beta-Amyloid (1-42), Human | Anaspec | AS-20276 | |
| Thrombin from human plasma | Sigma-Aldrich | T7009 | |
| 1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99% | Sigma-Aldrich | 105228 | |
| DIMETHYL SULFOXIDE (DMSO), STERILE-FILTERED | Sigma-Aldrich | D2438 | |
| Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
| Tris Base | Fischer Scientific | BP152 | |
| HEPES | Fischer Scientific | BP310 | |
| NaCl | Fischer Scientific | S271 | |
| CaCl2 | Fischer Scientific | C70 | |
| Filter Syringe, 0.2µM, 25mm | Pall | 4612 | |
| Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia. | Millipore | SLVV033RS | |
| Solid 96 Well Plates High Binding Certified Flat Bottom | Fischer Scientific | 21377203 | |
| Spectramax Plus384 | Molecular Devices | 89212-396 | |
| Centrifuge, 5417R | Eppendorf | 5417R | |
| Branson 200 Ultrasonic Cleaner | Fischer Scientific | 15-337-22 | |
| Lab Rotator | Thermo Scientific | 2314-1CEQ | |
| Spare washers for cover slip holder | Tousimis | 8766-01 | |
| Narrow Stem Pipets – Sedi-Pet | Electron Microscopy Sciences (EMS) | 70967-13 | |
| Sample holder for CPD (Cover slip holder) | Tousimis | 8766 | |
| Mount Holder Box, Pin Type | Electron Microscopy Sciences (EMS) | 76610 | |
| Round glass cover slides (12 mm) | Hampton Research | HR3-277 | |
| 10% Glutaraldehyde | Electron Microscopy Sciences (EMS) | 16120 | |
| Ethanol | Decon Labs | 11652 | |
| 24 well plate | Falcon | 3047 | |
| Na Cacodylate | Electron Microscopy Sciences (EMS) | 11652 | |
| SEM Stubs, Tapered end pin. | Electron Microscopy Sciences (EMS) | 75192 | |
| PELCO Tabs, Carbon Conductive Tabs, 12mm OD | Ted Pella | 16084-1 | |
| Autosamdri-815 Critical Point Dryer with Gold/Palladium target | Tousimis | ||
| Denton Desk IV Coater | Denton Vacuum | ||
| Leo 1550 FE-SEM | Carl Zeiss | ||
| Smart SEM Software | Carl Zeiss |
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