猪外周血血液生长内皮细胞（BOEC）的表征
Abstract
内皮是一种动态的综合结构，在血管生成、止血、炎症和体内平衡等许多生理功能中起重要作用。内皮在动脉粥样硬化、高血压和糖尿病等病理生理学中也起着重要作用。内皮细胞形成血液和淋巴管的内层，在结构和功能上表现出异质性。各种小组已经评估了来源于人外周血的内皮细胞的功能，重点是来源于造血干细胞或成熟血液生长内皮细胞（或内皮集落形成细胞）的内皮祖细胞。这些细胞为治疗和疾病建模提供了自体资源。异种细胞可以提供另一种治疗来源，因为它们的可用性和同质性通过使用在类似条件下饲养的遗传相似的动物来实现。因此，已经提出了一种从猪外周血中分离和扩增高度增殖的血液生长内皮细胞的稳健方案。这些细胞可用于多种应用，如心血管组织工程、细胞治疗、疾病建模、药物筛选、研究内皮细胞生物学以及 体外 共培养，以研究异种移植中的炎症和凝血反应。
Introduction
内皮是一种高度复杂的动态结构，是血管壁的重要组成部分。它排列在血管的内表面，在循环血液和周围组织之间提供物理界面。已知这种异质结构执行各种功能，例如血管生成，炎症，血管调节和止血1，2，3，4。人脐静脉内皮细胞是一种被广泛研究的细胞类型，用于评估内皮细胞的功能。然而，患者特异性批次变异性、表型不一致和最小分裂效率表明需要确定可以改善所有这些特征的细胞来源5。
获得原代内皮细胞的均质群体在技术上可能具有挑战性，并且原代内皮细胞不具有高增殖能力6。因此，为了研究血管再生和评估病理生理过程，各个小组试图获得和评估来自外周血的不同类型的内皮细胞，例如内皮祖细胞（EPC）或血液生长内皮细胞（BOEC）6，7，8，9.纺锤形的早期EPC起源于造血干细胞（HSC），具有有限的生长效力和有限的血管生成能力，无法产生成熟的内皮细胞。此外，它们与炎性单核细胞非常相似。此外，它们进一步分化成功能性、增殖、成熟的内皮细胞的能力仍有争议6，7，9，10。外周血单核细胞 （PBMC） 的连续培养可产生称为晚期生长 EPC、BOEC 或内皮集落形成细胞 （ECFC） 的二次细胞群6，7，9，10。Medina等人在2018年承认EPC的局限性，其命名的模糊性，以及与EPC11下连续分组的许多不同细胞类型普遍缺乏一致性。相比之下，BOEC因其在血管修复，健康和疾病以及细胞治疗中的作用而得到认可。这些细胞的进一步研究和治疗使用将依赖于方案，以一致地从循环祖细胞中获取这些细胞类型。
BOEC等原代细胞可用作替代细胞，以获得高度增殖的成熟内皮细胞6。BOEC在表型上与早期EPC不同，并表现出典型的内皮特征，例如鹅卵石形态以及粘附连接和洞穴的表达12。Hebbel等人的基因分析13，14，15发现BOEC或ECFC是真正的内皮细胞，因为它们促进微血管和大血管的形成。因此，BOEC可以用作评估病理生理过程和遗传变异的工具16。它们也被认为是血管再生细胞疗法的极好细胞来源17。因此，一个标准化的方案来持续获得这些高度增殖的细胞是必不可少的。
虽然BOEC为研究人类病理生理和遗传变异提供了强大的工具，但更同质的BOEC来源可能会提供更强大和可靠的实验和治疗结果。通过使用来自在相似条件下饲养的遗传相似动物的异种细胞源，可以实现卓越的同质性18。虽然异种细胞来源容易引发宿主免疫反应，但正在开发免疫调节策略，目的是产生免疫相容的动物和动物产品，包括细胞。特别是猪，是外周血的丰富来源，由于与人类的解剖和生理相似性，通常用于研究医疗设备和其他疗法。因此，本研究完善了从猪外周血中分离和扩增高度增殖的BOEC的方案。下面详述的方案是一种从相对少量的血液中获取大量 BOEC 的简单可靠的方法。培养物可以通过多次传代扩增，以从单个血液样本中产生数百万个细胞。
Protocol
所有动物研究均由威斯康星医学院和妙佑医疗国际各自的机构动物护理和使用委员会 （IACUC） 批准。 注意：在这项研究中，使用了约克郡/兰德雷斯/杜洛克杂交家猪（Sus homeus），雄性和雌性，40-80公斤，3-6个月大。 1.猪外周血采集 准备材料。在无菌盐水中将肝素溶液稀释至100U / mL。 向两个 50 mL 锥形管和两个 60 mL…
Representative Results
从培养开始到观察到BOEC菌落，观察培养细胞的形态（图1）。较小的贴壁细胞群开始附着在培养皿上并生长，而非贴壁细胞随着培养基的变化而被移除（图1B）。菌落在第6天首次出现，作为从中心点向外放射状增殖的内皮样细胞的集合（图1D）。随着培养的进行，细胞集落变得更加致密，并显示出类似于成熟内皮细胞的鹅卵石形态…
Discussion
BOEC是一种强大的工具，可用于各种科学和治疗方法7，8，16。BOEC已被用于分析EC基因表达，以阐明导致血管疾病和癌症发展的关键因素5，19，20，21。BOEC还被用于治疗应用，如血管再生和基因传递22，<sup cl…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者希望感谢NIH / NHLBI R00 HL129068的资助。
Materials
| 19 G needle | Covidien | 1188818112 | |
| 50 mL conical tubes | Corning | 352098 | |
| 6 well plate | BD Falcon | 353046 | |
| 60 mL syringes | Covidien | 8881560125 | |
| Ammonium chloride solution (0.8%) | Stemcell Technologies | 07850 | |
| Antibiotic/antimycotic solution (100x) | Gibco | 15240-062 | |
| Centrifuge | Thermo Scientific | 75-253-839 | |
| EGM-2 culture medium | Lonza Walkersville | CC-3162 | |
| Extension tube | Hanna Pharmaceutical Supply Co. | 03382C6227 | |
| Fetal bovine serum (FBS) | Atlas Biologicals | F-0500-A | |
| Ficoll-Paque 1077 | Cytiva | 17144003 | Density gradient solution |
| Heparin sodium injection (1,000 units/mL) | Pfizer | 00069-0058-01 | |
| Human plasma fibronectin | Gibco | 33016-015 | |
| Ice | N/A | N/A | |
| Phosphate-buffered saline (PBS) | Gibco | 10010-023 | |
| Pipette set | Eppendorf | 2231300004 | |
| Sterile water | Gibco | 15230-162 | |
| Thin pipette | Celltreat Scientific | 229280 |
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Citar este artigo
Shradhanjali, A., Uthamaraj, S., Dragomir-Daescu, D., Gulati, R., Sandhu, G. S., Tefft, B. J. Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood. J. Vis. Exp. (179), e63285, doi:10.3791/63285 (2022).