基质凝胶变化对微血管内皮细胞原代培养功能的影响

IF 1.9 4区 医学 Q3 HEMATOLOGY
Pía C. Burboa, Juliana C. Corrêa-Velloso, Cecilia Arriagada, Andrew P. Thomas, Walter N. Durán, Mauricio A. Lillo
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引用次数: 0

摘要

目的:内皮调节血管功能的重要方面,包括止血、血管运动张力、增殖、免疫细胞粘附和微血管通透性。内皮细胞(EC),尤其是动脉血管中的内皮细胞,对血流分布和外周阻力调节起着关键作用。研究血管内皮生理学,尤其是微血管内皮细胞,需要精确的分离和培养技术:方法:新鲜分离的血管内皮细胞对于研究蛋白质表达、离子通道行为和钙动力学至关重要。建立原代内皮细胞培养对揭示血管功能和了解完整微血管内皮的作用至关重要。尽管意义重大,但在微血管研究中,详细的方案和与完整血管的比较却很少见。我们开发了一种可重复的方法来分离微血管内皮细胞,通过在纤维连接蛋白和明胶基质凝胶上培养细胞来评估基质的影响。这种比较方法增强了我们对微血管内皮细胞生物学的了解:结果:微血管系膜内皮细胞在两种基质凝胶上都表达了关键标记(VE-cadherin 和 eNOS),证实了细胞培养的纯度。在无涂层条件下,未检测到心血管细胞,而与平滑肌细胞和成纤维细胞相关的蛋白质却很明显。研究不同基质基底上内皮细胞(EC)的生理动态发现,明胶和纤连蛋白基质凝胶上的雌雄内皮细胞的细胞长度、形状和 Ca2+ 升高具有可比性。明胶培养的心肌对乙酰胆碱(ACh)或三磷酸腺苷(ATP)表现出类似的膜电位反应,这与纤维粘连蛋白培养的心肌形成鲜明对比。在没有刺激的情况下,纤连蛋白培养的心肌比明胶培养的心肌显示出更多的去极化静息膜电位:明胶培养的内皮细胞表现出与小鼠肠系膜动脉完整内皮细胞相似的电行为,从而加深了我们对内皮细胞在不同微环境中行为的理解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function

Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function

Objective

The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques.

Methods

Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology.

Results

Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident.

Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs.

Conclusions

Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.

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来源期刊
Microcirculation
Microcirculation 医学-外周血管病
CiteScore
5.00
自引率
4.20%
发文量
43
审稿时长
6-12 weeks
期刊介绍: The journal features original contributions that are the result of investigations contributing significant new information relating to the vascular and lymphatic microcirculation addressed at the intact animal, organ, cellular, or molecular level. Papers describe applications of the methods of physiology, biophysics, bioengineering, genetics, cell biology, biochemistry, and molecular biology to problems in microcirculation. Microcirculation also publishes state-of-the-art reviews that address frontier areas or new advances in technology in the fields of microcirculatory disease and function. Specific areas of interest include: Angiogenesis, growth and remodeling; Transport and exchange of gasses and solutes; Rheology and biorheology; Endothelial cell biology and metabolism; Interactions between endothelium, smooth muscle, parenchymal cells, leukocytes and platelets; Regulation of vasomotor tone; and Microvascular structures, imaging and morphometry. Papers also describe innovations in experimental techniques and instrumentation for studying all aspects of microcirculatory structure and function.
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