人隐静脉内皮细胞分离和暴露于受控水平的剪切应力和拉伸
Summary
我们描述了一种分离和培养人隐静脉内皮细胞（hSVEC）的方案。我们还提供了产生剪切应力和拉伸的详细方法，以研究hSVEC中的机械应力。
Abstract
冠状动脉旁路移植术（CABG）是一种对缺血性心肌进行血运重建的手术。尽管与动脉导管相比，长期通畅性降低，但隐静脉仍用作冠状动脉旁路移植术导管。与移植物动脉化相关的血流动力学应激突然增加导致血管损伤，尤其是内皮损伤，这可能会影响隐静脉移植物 （SVG） 的低通畅性。在这里，我们描述了人隐静脉内皮细胞（hSVEC）的分离，表征和扩增。通过胶原酶消化分离的细胞显示出典型的鹅卵石形态，并表达内皮细胞标志物CD31和VE-钙粘蛋白。为了评估机械应力的影响，本研究使用协议来研究动脉化SVG上的两种主要物理刺激，剪切应力和拉伸。 hSVEC在平行板流动室中培养以产生剪切应力，显示流动方向的对齐和KLF2，KLF4和NOS3的表达增加。hSVEC也可以在硅膜中培养，该硅膜允许模拟静脉（低）和动脉（高）拉伸的受控细胞拉伸。内皮细胞的F-肌动蛋白模式和一氧化氮（NO）分泌通过动脉拉伸相应地调节。总之，我们提出了一种分离hSVEC的详细方法，以研究血流动力学机械应激对内皮表型的影响。
Introduction
内皮细胞 （EC） 功能障碍是隐静脉移植失败的关键因素1，2，3，4。剪切应力和周期性拉伸的持续增加诱导人隐静脉内皮细胞（hSVEC）的促炎表型3，4，5，6。潜在的分子途径仍未完全了解，体外研究的标准化方案可能会利用该领域的新见解。在这里，我们描述了一个简单的方案来分离，表征和扩增hSVEC，以及如何将它们暴露于不同水平的剪切应力和循环拉伸，模拟静脉和动脉血流动力学状况。
hSVEC通过胶原酶孵育分离，可使用至第8代。与其他可用的方案7相比，该协议需要较少的血管操作，从而减少了平滑肌细胞和成纤维细胞的污染。另一方面，它需要至少 2 厘米的较大容器段才能进行有效的 EC 提取。在文献中，已经报道了来自大型容器的EC也可以通过机械去除获得7，8。虽然有效，但物理方法具有EC产量低和成纤维细胞污染较高的缺点。为了提高纯度，需要使用磁珠或细胞分选的额外步骤，由于获取磁珠和抗体7，8，增加了方案的成本。酶法在EC纯度和活力方面具有更快更好的结果7，8。
研究内皮功能障碍最常用的EC是人脐静脉内皮细胞（HUVECs）。众所周知，EC表型在不同的血管床中发生变化，因此必须开发代表所研究血管的方法9，10。在这方面，建立分离hSVEC并在机械应力下培养的方案是了解hSVEC功能障碍在静脉移植疾病中的贡献的宝贵工具。
Protocol
未使用的隐静脉段是从圣保罗大学医学院心脏研究所（InCor）接受主动脉冠状动脉搭桥手术的患者那里获得的。所有个体都知情同意参与该研究，该研究由当地伦理委员会审查和批准。 1. 原代人隐静脉内皮细胞 （hSVEC） 的分离、培养和表征 制备高压灭菌一对直镊子或弯曲的镊子和组织剪刀（7-8厘米）。 准备无菌明胶。将0.1g（0.1%w / v）或3g?…
Representative Results
通常，粘附的EC可以在提取后3-4天观察到。hSVEC最初形成细胞簇并显示出典型的“鹅卵石”形态（图1B）。它们表达EC标志物CD31（图1C，D）和VE-钙粘蛋白（图1D）。hSVEC可以在非包被处理的细胞培养皿上轻松繁殖，并且在培养物中保留内皮表型多达八代。 hSVEC在剪切应力下培养时，沿流动方向对齐（…
Discussion
隐静脉段应至少为 2 cm，以成功分离 hSVEC。小段难以处理并绑住血管末端以维持胶原酶溶液以分离细胞。减小的管腔表面积不能产生足够的细胞来扩增培养物。为了尽量减少非EC污染的风险，在整个过程中，大隐静脉段的操作需要非常轻柔。将移液器吸头引入管腔表面以去除血液和引入胶原酶溶液时要小心。应很好地控制酶溶液的暴露（不超过1小时），以减少非EC对培养物的污染。使用补充生长因…
Declarações
The authors have nothing to disclose.
Acknowledgements
JEK得到了圣保罗国家宪法权利保护基金会[FAPESP-INCT-20214/50889-7和2013/17368-0]和全国发展委员会（INCT-465586/2014-7和309179/2013-0）的资助。AAM得到了圣保罗国家宪法权利保护基金会（FAPESP 2015/11139-5）和全国公民和技术发展委员会（环球-407911/2021-9）的资助。
Materials
| 0.25% Trypsin-0.02% EDTA solution | Gibco | 25200072 | |
| 15 µ slide I 0.4 Luer | Ibidi | 80176 | |
| 4',6-Diamidino-2-Phenylindole, Dilactate (DAPI) | Thermo Fisher Scientific | D3571 | |
| 6-wells equibiaxial loading station of 25 mm | Flexcell International Corporation | LS-3000B25.VJW | |
| 8-well chamber slide with removable well | Thermo Fisher Scientific | 154453 | |
| Acetic Acid (Glacial) | Millipore | 100063 | |
| Acrylic sheet 1 cm thick | Plexiglass | ||
| Anti-CD31 antibody | Abcam | ab24590 | |
| Anti-CD31, FITC antibody | Thermo Fisher Scientific | MHCD3101 | |
| Anti-VE-cadherin antibody | Cell Signaling | 2500 | |
| Bioflex plates collagen I | Flexcell International Corporation | BF3001C | |
| Bovine serum albumin solution | Sigma-Aldrich | A8412 | |
| Cotton suture EP 3.5 15 x 45 cm | Brasuture | AP524 | |
| Cyclophilin forward primer | Thermo Fisher Scientific | Custom designed | |
| Cyclophilin reverse primer | Thermo Fisher Scientific | Custom designed | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D4540 | |
| EBM-2 basal medium | Lonza | CC3156 | |
| EGM-2 SingleQuots supplements | Lonza | CC4176 | |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific | 2657-029 | |
| Flexcell FX-5000 tension system | Flexcell International Corporation | FX-5000T | |
| Fluoromount aqueous mounting medium | Sigma-Aldrich | F4680 | |
| Gelatin from porcine skin | Sigma-Aldrich | G2500 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Goat anti-Mouse IgG Alexa Fluor 488 | Thermo Fisher Scientific | A11001 | |
| Goat anti-Rabbit IgG Alexa Fluor 488 | Thermo Fisher Scientific | A11008 | |
| Heparin sodium from porcine intestinal mucosa 5000 IU/mL | Blau Farmacêutica | SKU 68027 | |
| Ibidi pump system (Pump + Fluidic Unit) | Ibidi | 10902 | |
| KLF2 forward primer | Thermo Fisher Scientific | Custom designed | |
| KLF2 reverse primer | Thermo Fisher Scientific | Custom designed | |
| KLF4 forward primer | Thermo Fisher Scientific | Custom designed | |
| KLF4 reverse primer | Thermo Fisher Scientific | Custom designed | |
| NOA 280 nitric oxide analyzer | Sievers Instruments | NOA-280i-1 | |
| NOS3 forward primer | Thermo Fisher Scientific | Custom designed | |
| NOS3 reverse primer | Thermo Fisher Scientific | Custom designed | |
| Paraformaldehyde (PFA) | Sigma-Aldrich | 158127 | |
| Perfusion set 15 cm, ID 1.6 mm, red, 10 mL reservoirs | Ibidi | 10962 | |
| Phalloidin – Alexa Fluor 488 | Thermo Fisher Scientific | A12379 | |
| Phalloidin – Alexa Fluor 568 | Thermo Fisher Scientific | A12380 | |
| Phosphate buffered saline (PBS), pH 7.4 | Thermo Fisher Scientific | 10010031 | |
| Potassium Iodide | Sigma-Aldrich | 221945 | |
| QuanTitec SYBR green PCR kit | Qiagen | 204143 | |
| QuantStudio 12K flex platform | Applied Biosystems | 4471087 | |
| RNeasy micro kit | Quiagen | 74004 | |
| Slide glass (24 mm x 60 mm) | Knittel Glass | VD12460Y1D.01 | |
| Sodium nitrite | Sigma-Aldrich | 31443 | |
| SuperScript IV first-strand synthesis system | Thermo Fisher Scientific | 18091200 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| Trypan blue stain 0.4% | Gibco | 15250-061 | |
| Type II collagenase from Clostridium histolyticum | Sigma-Aldrich | C6885 |
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Citar este artigo
Girão-Silva, T., Fonseca-Alaniz, M. H., Oliveira Dallan, L. A., Valãdao, I. C., Oliveira da Rocha, G. H., Krieger, J. E., Miyakawa, A. A. Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch. J. Vis. Exp. (194), e65122, doi:10.3791/65122 (2023).