A general strategy for isolation of endothelial cells from murine tissues - Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants
A general strategy for isolation of endothelial cells from murine tissues - Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants
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DOI：
10.1161/01.atv.17.8.1599
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发表时间：
1997-08-01
影响因子：
8.7
通讯作者：
Vecchi, A
中科院分区：
文献类型：
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作者：
Dong, QG;Bernasconi, S;Vecchi, A
一种快速、可重复的鼠内皮细胞 (EC) 分离方法已被开发出来。通过胶原酶消化机械切碎的肺和皮下海绵植入物，然后用大鼠抗小鼠 CD31（即 PECAM-1）单克隆抗体包被的磁珠（Dynabeads）进行特异性选择，高度富集小鼠 EC。通过第二轮免疫磁性选择从原代培养物中分离出纯 EC 群体。然后通过有限稀释方法克隆来自肺的细胞，以排除非内皮细胞污染的可能性。在铺板的 300 个细胞中，获得了 29 个克隆（约 10%）。通过流式细胞仪测量，这些克隆呈 CD31 阳性，然后将来自肺部的 1 个克隆 (1G11) 和来自海绵植入物的细胞 (称为 SIEC) 分别进行后续体外培养 40 和 30 代（长达 5 个月）。对第 3 代和第 10 代之间的细胞进行表征。两种细胞类型均在明胶上形成接触抑制单层，并在基质胶上形成毛细管状“管”。然而，1G11 细胞表现出“鹅卵石”形态，而 SIEC 在汇合时具有成纤维细胞样外观。通过流式细胞术和酶联免疫吸附测定，这些细胞组成型表达CD31、VE-cadherin (cadherin-5)、CD34、ICAM-1、VCAM-1和P-选择素。用 30 ng/mL 肿瘤坏死因子-α 刺激后，细胞 E-选择素呈阳性（刺激后 4 小时），并且 ICAM-1、VCAM-1 和 P-选择素的表达上调（刺激 24 小时后）。通过荧光显微镜和 Northern blot 分析证实了 1G11 细胞和 SIEC 中 VE-钙粘蛋白的存在。两种细胞类型的表型和形态在培养 5 个月期间保持稳定，并且没有污染细胞过度生长的证据。综上所述，本文概述的方法可以为从多种鼠科组织中分离和培养 EC 提供一般策略。这里概述的总体策略简单、有效且灵活，允许包含进一步的正向或负向选择步骤。
A rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (ie, PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (approximate to 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like ''tubes'' on Matrigel. However, 1G11 cells exhibited a ''cobblestone'' morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.