通过CD47衍生肽固定，缓解血系细胞附着到金属植入物
Summary
此处介绍的是使用多磷酸盐化学将肽 CD47 （pepCD47） 附在金属支架上的规程。使用 pepCD47 使金属支架功能化可防止炎症细胞的附着和激活，从而提高其生物相容性。
Abstract
与裸金属支架和药物椭圆支架相关的主要并发症分别是支架内性恢复和后期支架血栓形成。因此，提高金属支架的生物相容性仍然是一个重大挑战。该协议的目标是描述一种由生物活性肽进行金属表面修饰的强有力技术，以提高血液接触医学植入物（包括血管内支架）的生物相容性。CD47是免疫学物种特异性自我标记，具有抗炎特性。研究表明，22种氨基酸肽与细胞外区域CD47的Ig域（pepCD47）相对应，具有抗炎特性，如全长蛋白。大鼠体内研究，以及我们实验室对兔子和人类血液实验系统的外体研究已经证明，在金属上进行pecd47固定通过防止炎症细胞附着和激活，提高了其生物相容性。本文介绍了金属表面和肽附着功能化的分步协议。使用具有潜在硫醇组（PABT）的聚酰胺双磷酸盐进行改性，然后通过与安装在苯丙胺组（PEI-PDT）的聚乙烯胺发生反应，对硫醇脱保和硫醇反应位点进行扩增。最后，pepCD47通过双8-3，6-二甲苯基分离基器将最终半胱氨酸残留物与核心肽序列连接起来，通过二硫化物键附着在金属表面。这种肽附着在金属表面的方法是有效的，相对便宜，因此可以应用于提高几种金属生物材料的生物相容性。
Introduction
皮下冠状动脉介入是治疗冠状动脉疾病（CAD）的第一线，主要涉及支架患病动脉。然而，支架内性休息（ISR）和支架血栓形成是常见的并发症与支架部署1。血-支架界面的血液相互作用的特点是几乎立即吸附金属表面的血浆蛋白，其次是血小板和炎症细胞附着和活化2。从活性炎症细胞释放炎症细胞因子和化疗素导致血管平滑肌细胞 （VSMC） 在 tunica 介质中的表型修饰，并触发其向心迁移到刺激室。活性VSMC在内膜中的扩散导致刺激层增厚，流明变窄和支架内休息3。药物吹风支架（DES）是为了防止VSMC扩散而开发的;然而，这些药物对内皮细胞4，5有非靶对目标细胞毒性作用。因此，后期支架血栓形成是一种常见的并发症与DES6，,7。由可生物降解聚合物（如聚L-乳酸酯）制成的支架在动物实验和初步临床试验中显示出了许多希望，但最终在”现实生活”的临床应用显示出其低于第三代DES8时被召回。因此，有必要提高裸金属支架的生物相容性，以更好的患者结果。
CD47是一种无处不在的表达跨膜蛋白，当与同源受体信号调节蛋白α（SIRP+）9结合时，抑制先天免疫反应。SIRP® 受体具有免疫细胞酪氨酸抑制因子 （ITIM） 域和 SIRP+ – CD47 相互作用时的信号事件最终导致炎症细胞活化10、 11,、12、13的调节。我们实验室的研究表明，重组CD47或其肽衍生物，对应于CD47（pepCD47）细胞外区域的22个氨基酸Ig域，可以降低宿主对一系列临床相关生物材料14、15、16,15的免疫反应。最近，我们已经证明peCD47可以固定在不锈钢支架表面，并显著减少与神经症相关的病理生理反应。值得注意的是，pepCD47改性表面可满足长期储存和环氧乙烷灭菌等相关使用条件。为此，pepCD47可能是一个有用的治疗目标，以解决血管内支架的临床局限性。
pepCD47与金属表面共价附着的策略涉及金属表面的一系列新颖的化学修饰。金属表面首先涂上具有潜在硫醇组 （PABT） 的聚酰胺双磷酸盐，然后对硫醇进行脱保，并附着已安装的二乙酰二硫磷酸组 （PDT）。在反应中未消耗的PET组与脱保护的PABT硫醇发生反应，然后用peCD47在终端半胱氨酸残留物中加入硫醇，从而通过二硫化物键,14、17、18,17将pecd47结合到金属表面。18我们使用荧光结合肽CD47（TAMRA-pepCD47）来确定肽的输入浓度，导致肽的最大表面固定。最后，我们分别评估了pecd47涂层金属表面的急性和慢性抗炎能力，使用钱德勒循环装置进行外体检测，以及单细胞附着/巨噬细胞扩张测定。
本文为硫化肽附着在金属表面提供了系统化方案;确定肽的最大固定密度;并评估pecd47涂层金属表面暴露在全血和孤立的单核细胞中的抗炎特性。
Protocol
Representative Results
Discussion
我们演示并描述了一种相对新颖的化学策略，将治疗性肽莫伊蒂斯附在不锈钢表面，其首要目标是减少表面与血液中的炎症细胞的活性。本文描述的双磷酸盐化学涉及PABT的金属氧化物和双磷酸盐组之间的协调键的形成。在金属表面形成的多磷酸单层厚度不超过5纳米18，因此，对厚聚合物涂层20的潜在刺激作用无关紧要。PABT的脂质侧链中潜在硫醇组的脱保护使金…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本文件提出的协议开发和研究得到了NIH（NBIB）R01基金（# EB023921）对国际基金和SJS的资助，NIH（NHLBI）R01基金（#HL137762）对国际基金和RJL的资助。
Materials
| 1 M Tris-HCL | Invitrogen | 15567-027 | pH – 7.5 |
| 4% Glutaraldehyde | Electron Microscopy Sciences | 16539-07 | |
| 4% Sodium Citrate | Sigma | S5770 | |
| ACK lysing buffer | Quality Biologicals | 118-156-721 | |
| anti-CD45RA Ab (mouse anti-rat; clone OX-19) | Biolegend | 202301 | |
| anti-CD5 Ab (mouse anti-rat; clone OX-19) | Biolegend | 203501 | |
| anti-CD6 Ab (mouse anti-rat; clone OX-52) | BD Biosciences | 550979 | |
| anti-CD68 Ab (mouse anti-rat; clone ED-1) | BioRad | MCA341 | |
| anti-CD8a Ab (mouse anti-rat; clone OX-8) | Biolegend | 201701 | |
| Chloroform Certified ACS | Fisher Chemical | C298-500 | |
| Dimethyl Formammide (DMF) | Alfa Aesar | 39117 | |
| Embra stainless steel grid | Electron Microscopy Sciences | E200-SS | stainless steel mesh mesh disks |
| Ficoll Hypaque | GE Healthcare | 17-1440-02 | |
| Glacial acetic acid | ACROS organic | 148930025 | |
| goat anti-mouse IgG Alexa Fluor | ThermoFisher | A11030 | |
| Heparin sodium | Sagent Pharmaceuticals | 402-01 | |
| Human pepCD47 | Bachem | 4099101 | |
| Isopropanol | Fisher Chemical | A426P-4 | |
| Metal adapters | Leur Fitting | 6515IND | 1 way adapter 316 ss 1/4"-5/16" hoes end |
| Methanol | RICCA chemical company | 4829-32 | |
| Microscope | Nikon Eclipse | TE300 | |
| Phosphate buffered saline (PBS) | Gibco | 14190-136 | |
| Pottasium Bicarbonate (KHCO3) | Fisher Chemical | P184-500 | |
| PVC tubes | Terumo-CVS | 60050 | 1/4" X 1/16 8' |
| sodium cacodylate buffer with 0.1M sodium chloride | Electron Microscopy Sciences | 11653 | |
| Sodium Dodecyl Sulfate (SDS) | Bio-Rad laboratories | 161-0302 | |
| Sodum actetate (C2H3NaO2) | Alfa Aesar | A13184 | |
| Src peptide | Bachem | 4092599 | |
| Stainless steel (AISI 304) cylinder-shaped samples with a lumen | Microgroup, Medway, MA | 20097328 | 1 cm X 6 mm OD |
| Stainless steel foils (AISI 316L) | Goodfellow, Coraopolis, PA | 100 mm X 100 mm X 0.05 mm | |
| Tetramethylrhodamine-conjugated pepCD47 (TAMRA-pepCD47) | Bachem | 4100277 | |
| TMB (3,3’ ,5,5’ -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) | Thermo Scientific | PG82089 | |
| Tween-20 | Bio-Rad laboratories | 170-6531 | |
| Vybrant CFDA SE Cell Tracer Kit | Invitrogen | V12883 |
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