微型猪原主主动脉内皮细胞的隔离与培养
1Department of Nephrology,Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 2Shenzhen Xenotransplantation Medical Engineering Research and Development Center,Shenzhen Second People’s Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, 3Department of Medical Laboratory,Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 4Department of Endocrinology,Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 5Xenotransplantation Program, Department of Surgery,University of Alabama at Birmingham, 6People’s Hospital of Longhua
Summary
介绍了一种从微型猪中分离原发猪主动脉内皮细胞(pAECs)的有效酶法。分离的初级pAEC可用于研究异种移植中的免疫和凝固反应。
Abstract
异种移植是解决终末期器官衰竭患者人体器官短缺的一种有希望的方法,猪被认为是合适的器官来源。免疫排斥和凝固是异种移植成功的两个主要障碍。血管内皮细胞(EC)损伤和功能障碍对异种移植中炎症和凝血反应的发展具有重要意义。因此,分离猪主动脉内皮细胞(pAECs)对于研究免疫排斥和凝固反应是必要的。在这里,我们开发了一种简单的酶法,用于从微型猪中分离、表征和扩展高度纯化的PAE。首先,微型猪被用氯胺酮麻醉,并切除了一条大头塔的长度。其次,主肠的内皮表面暴露于0.005%胶原酶IV消化溶液15分钟。 第三,主肠的内皮表面只刮一个方向与细胞刮刀(<10次),并没有压缩过程中刮。最后,通过流式细胞测定与抗CD31抗体测定第3天和通道1和通道2之后的分离的PAEC。CD31阳性细胞的百分比分别为97.4%~1.2%、94.4%~1.1%和92.4%~1.7%(均值=SD)。胶原酶IV的浓度、消化时间、方向、刮擦的频率和强度对于减少成纤维细胞污染、获得高纯度和大量EC至关重要。总之,我们的酶法是一种从微型猪主主塔中分离ECs的高效方法,细胞可以在体外扩展,以研究异种移植中的免疫和凝固反应。
Introduction
移植器官短缺是一个突出的世界性问题。根据中国红十字会的数据,2018年,中国只有少数期器官衰竭患者获得了合适的器官。
异体移植是解决器官短缺问题的一种有希望的途径。猪器官被认为是最适合人类的器官,因为解剖学和生理上的相似性2,3。猪异种移植的失败很大程度上与灵长类动物的免疫排斥和凝固反应有关。猪内皮细胞(ECs)至关重要,因为这些细胞是第一个与灵长类免疫系统相互作用的细胞,包括抗体、补体、细胞因子和免疫细胞(例如T细胞、B细胞和巨噬细胞)4、5。猪EC在猪器官和小岛异种移植中起着至关重要的作用。因此,ECs是研究猪移植物的排斥和凝固反应的重要细胞。异种移植研究需要分离高质量的猪E。
在试图从不同的器官(例如心脏、肾脏、肝脏和大头塔)中分离EC时,在异种移植设置8、9、10、11、12中报告了几种方案。然而,保持隔离的EC的超纯文化是标准协议中的一个突出问题。在目前的研究中,消化溶液浓度增加、消化时间和刮削强度不当,可能导致成纤维细胞污染增加。此外,对微型猪的分离方法的研究较少。在这里,我们描述了一种优化的酶法,从微型猪(武志山或巴马)中分离出高度纯化的PAE。该协议中的几个步骤已经设计用于减少成纤维细胞污染和获得高纯度的ECs。
Protocol
动物的使用是深圳市第二人民医院伦理审查委员会根据动物福利原则批准的。 1. 动物、介质和缓冲液的准备 准备微型猪。注:所有微型猪都是中国五指山或巴马猪(雄性)。猪的年龄和体重分别为100天~8天和5.7公斤=1.0公斤(均值=SD,n = 3)。 准备培养基:内皮细胞培养基(ECM)辅以10%(v/v)热灭活胎儿牛血清(FBS),1%(v/v)青霉素/链霉素(P/S)和1%(v/v)内皮细胞生长补充剂(ECGS)。</l…
Representative Results
我们的方法旨在提供一种有效的方法,从主头塔中分离出高度纯化的E从微型猪。主数手术的过程如图1所示。第一步是从猪中切除了整个主塔。防止其他细胞或细菌污染是非常重要的。因此,不要伤害其他器官或组织,以防非靶向细胞或细菌污染主方,用预冷却洗涤缓冲液3x清洗主塔(图1B-D)。保持细胞活力的另一个关键步骤是尽量减少从获得手术…
Discussion
内皮细胞通常用于血管功能障碍、糖尿病、组织再生、移植和癌症14、15、16、17、18的研究。为了了解和描述EC在这些疾病中的生物学和功能,已经报道了许多方法分离不同患病器官或组织的EC8,19,20<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了广东省自然科学基金的资助,资助/奖励编号:2016A030313028;广东省医学科学研究基金会,资助/奖励编号:B2018003;深圳市科技基金会,授予/奖励编号:JCYJ20180306172449376, JCYJ20180306172459580, JCJY2016022292204849975, GJHZ2017031417171357556, JCYJ20160103000011 和 JCYJ2016016042822040404444;深圳市龙华区科技基金会,授奖/奖励编号:2017013;国家重点研发项目,授权/奖励编号:2017YFC1103704;深圳三明医药项目,授予/奖励编号:SZSM201412020;广东省高水平医院建设专项资金(2019年);深圳市高水平医学学科建设基金,助学金/奖励号:2016031638;深圳市卫生和计划生育委员会,授予/奖励编号:SZXJ2017021和SZXJ2018059。我们感谢深圳大学的张汉成和赵志成协助编写手稿。
Materials
| BD FACSAria II | BD Bioscience | ||
| Boneforceps | Beijing HeLi KeChuang Technology Development CO.,Ltd. China | HL-YGQ | |
| BOON Disposable Syringe (10ml) | Jiangsu Yile medical Article Co., Ltd. China | ||
| CD31-FITC antibody | Bio-Rad | MCA1746F | |
| Cell scraper | Corning | 3010# | |
| Collagenase IV | Sigma-Aldrich | C5138#-1G | |
| Compound ketamine injection | Veterinary Pharmaceutical Factory of Shenyang, China | Ketamine Hydrochloride：0.3g/2ml,Xylazine hydrochloride:0.3g/2ml, Phenacetin hydrochloride：1mg/2ml | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Life Technologies | D1306# | |
| DMEM | Life Technologies | 11965118# | |
| ECM | Sciencell | 1001# | |
| ECGS | Sciencell | 1052# | |
| Eppendorf Snap-Cap Microtube(1.5mL) | AXYGEN | MCT-150-C# | |
| Falcon 100mm Cell Culture Dish | Corning | 353003# | |
| Fetal Bovine Serum | GIBCO | 10270-106# | |
| Flowjo v10.0 | |||
| Forceps | ShangHai medical instruments Co.,Ltd.China | ||
| Heparin sodium | Jiangsu WanBang biopharmaceutical Co.,Ltd.China | ||
| Iodine tincture | Guilin LiFeng Medical Supplies Co.,Ltd.China | ||
| Miniature Pig (Bama or Wuzhishan) | Kang Yi Ecological Agriculture Co., Ltd, China | ||
| Mshot microscope | Guangzhou Micro-shot Technology Co., Ltd. | M152 | |
| Petri Dishes (150 x 15 mm) | Biologixgroup | 66-1515# | |
| Penicillin/Streptomycin | Life Technologies | 15070063# | |
| Rectangular Canted Neck Cell Culture Flask with Vent Cap （T25） | Corning | 3289# | |
| Scissors | ShangHai medical instruments Co.,Ltd.China | ||
| Serological Transfer Pipettes (10ml) | JET Biofil | GSP010010# | |
| Sterile Pasteur Pipette | GeneBrick | GY0025# | |
| Sterile Syringe Filter (0.22µm) | Millipore | SLGV033RS# | |
| Surgical scalpel | ShangHai medical instruments Co.,Ltd.China | 22# | |
| Surgical suture | Shanghai Pudong Jinhuan Medical Supplies Co., Ltd | 5-0# | |
| Syringe（5mL） | Shengguang Medical Instrument Co., Ltd.China | ||
| Trypsin-EDTA (0.25%), phenol red | GIBCO | 25200056# | |
| 75% Medical alcohol | Guilin LiFeng Medical Supplies Co.,Ltd.China | ||
| 20 x PBS solution (pH 7.4,Nuclease free) | Sangon Biotech | B540627# | |
| Medical disinfectant 84 liquid | Sichuan Province Yijieshi Medical Technology Co., Ltd | 450ml/bottle |
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Cite This Article
Zhao, Y., Zhao, C., Cooper, D. K., Lu, Y., Luo, K., Wang, H., Chen, P., Zeng, C., Luan, S., Mou, L., Gao, H. Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs. J. Vis. Exp. (150), e59673, doi:10.3791/59673 (2019).