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Biologia

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

Published: June 03, 2019
doi:
10.3791/59688
, , , , , , ,
1The State Key Laboratory of Pharmaceutical Biotechnology and the Department of Pharmacology and Pharmacy,University of Hong Kong

Summary

该协议描述了使用线状成像来评估从小鼠分离肠系膜动脉的神经间等距张力, 特别考虑的是内皮细胞和血管周围脂肪组织释放的因素的调节。

Abstract

血管张力改变对病理生理刺激的反应有助于广泛的心血管和代谢疾病的发展。内皮功能障碍是减少血管舒张和增强动脉血管收缩的主要元凶。动脉周围的脂肪组织在调节血管平滑肌细胞内皮依赖性松弛和/或收缩方面发挥着重要作用。内皮和血管周围脂肪组织之间的交叉谈话可以通过线断层成像系统在体内使用安装的血管进行评估。然而, 应建立从不同物种、年龄、遗传背景和/或病理生理条件的动物中获得的动脉的最佳设置。

Introduction

动脉扩张和收缩分别是通过血管平滑肌细胞的松弛和收缩来实现的。小动脉血管反应性的变化有助于自主神经和血液中存在的激素 (如儿茶酚胺、血管紧张素 II.、血清素、加压素) 对动脉血压的稳态调节。在局部一级, 平滑肌细胞的血管反应被来自内膜内皮细胞和动脉周围脂肪组织的信号所调节 (图 1)。

内皮不仅是一个被动屏障, 而且还作为一个表面, 以交换血液和潜在的血管平滑肌细胞之间的信号。通过释放各种血管活性物质, 内皮细胞在血管张力反应1的局部控制中起着至关重要的作用。例如, 在对乙酰胆碱的反应中, 内皮一氧化氮合酶 (eNOS) 在内皮细胞中被激活, 产生一氧化氮 (NO), 通过激活可溶性利尼环化酶 (sGC)诱导下血管平滑肌的松弛。2. 其他血管活性物质包括环氧合酶 (如前列腺素和血栓素 a2)、脂氧合酶 (如 12-羟基磷酸四烯酸、12-hete) 和细胞色素 p450 单胞酶 (hetes 和环氧血尿酸、Eet)、活性氧种类 (ros) 和血管活性肽 (如内皮素-1 和血管紧张素 II.) 和内皮衍生的超极化因子 (EDHF)3。内皮衍生血管扩张剂和血管收缩剂之间的微妙平衡保持局部血管运动张力4,5。

内皮功能障碍的特点是内皮依赖性血管舒张6的损伤, 血管老化的标志 7.随着年龄的发展, 内皮促进血管舒张的能力逐渐降低, 主要原因是 NO 生物利用度下降, 以及血管平滑肌细胞内皮细胞和 sGC 中 eNOS 的异常表达和功能8,9,10. no 的生物利用度降低, 可促进内皮依赖性血管收缩剂11,12的产生。在老年动脉中, 内皮功能障碍会导致介质增生, 这反映在壁厚、内侧细胞核数量的显著增加上, 这让人想起了高血压和动脉粥样硬化中观察到的动脉增厚。病人13,14。此外, 肥胖、糖尿病或高血压等病理生理条件加速内皮功能障碍的发展15,16。

血管周围脂肪组织 (PVAT) 释放大量脂肪因子, 以调节血管结构和功能17。Pvat 的抗收缩作用是由放松因子介导的, 如脂联素、no、过氧化氢和硫化氢 18,19, 20。然而, 根据位置和病理生理条件, PVAT 也可以提高不同动脉的收缩反应21。Pvat 产生的亲收缩物质包括血管紧张素 ii、瘦素、抵抗素和 ros22、23。 在大多数关于孤立血管的研究中, PVAT 被认为是血管的简单结构支撑, 因此在准备血管环段的过程中被切除。由于脂肪功能障碍是高血压和相关心血管并发症的一个独立的危险因素24,在调查血管的血管反应性时, 应考虑血管周围的 PVAT。不同的动脉。

多线肌瘤系统已被广泛用于研究各种血管的血管运动功能, 包括主动脉、肠系膜、肾、股、脑和冠状动脉 25,26。本文描述的协议将使用线断层成像来评估从转基因小鼠模型中分离出的肠系膜动脉中的血管反应性, 并特别关注 PVAT 的调节。

Protocol

所有用于以下研究的动物均由香港大学医学院实验动物组提供。获得了省实验室动物用于教学和研究委员会的道德认可 (CULATR, 编号: 4085-16)。 1. 准备工作 药物的制备 按照材料安全数据表 (MSDS) 中的规定, 在收到药物后立即妥善储存药物。将粉末形式的药物作为高浓度库存溶液溶解在溶剂中, 然后在-20°c 下储存。注: 大多数药物溶解在蒸馏水中, 以…

Representative Results

检查纵向/张力关系, 以获得归一化因子k 应用于血管段的拉伸量会影响阿克林-肌球蛋白相互作用的程度, 从而产生最大的作用力。因此, 对于每一种类型的血管, 确定最大活跃力所需的拉伸量是适当的脊髓造影研究所必需的。在这里, 对从小鼠模型中分离出的肠系膜动脉进行了纵向张力关系的归一化 (<strong clas…

Discussion

除了内皮细胞外, PVAT 产生的信号在调节平滑肌张力反应性 30方面发挥着重要作用。健康的 pvat 释放 no 和抗炎脂联素, 对动脉产生抗收缩作用, 在肥胖和代谢综合征等病理条件下丢失 31,32。在疾病状态下, pvat 有助于内皮功能障碍和其他心血管异常的发展33,34。据报道, 肥胖动物的动脉<sup class="x…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了香港研究赠款委员会 [17124718 和 17124718]、香港卫生和医学研究基金 [13142651 和 13142651]、香港合作研究基金 [C7055-14G] 和国家基金会的资助。中国研究项目 [973 项目 2015CB553603]。

Materials

Acetylcholine Sigma-Aldrich A6625 Stock concentration: 10-1 M
Working concentration: 10-10 to 10-5 M
L-NAME (Nω-nitro-L-arginine methyl ester) Sigma-Aldrich N5751 Stock concentration: 3 x 10-2 M
Working concentration: 10-4 M
Phenylephrine Sigma-Aldrich P6126 Stock concentration: 10-2 M
Working concentration: 10-10 to 10-5 M
U46619 (9,11-dideoxy-9α,11αmethanoepoxy prostaglandin F2α) Enzo BML-PG023-0001 Stock concentration: 10-5 M
Working concentration: 1-3 x 10-8 M
Multiwire myograph Danish MyoTechnology (DMT) 620M
PowerLab 4/26 ADInstruments ML848
Labchart7 ADInstruments –
Adipo-SIRT1 wild type mice Laboratory Animal Unit, The University of Hong Kong CULATR NO.: 4085-16
Silicon-coated Petri dishes Danish MyoTechnology (DMT)
Tungsten wires Danish MyoTechnology (DMT) 300331
Surgical tools
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Tags

Keywords: Vascular ToneMesenteric ArteriesIsometric ConditionVascular ResponsivenessHypertensionCardiovascular ComplicationsMyographNormalizationPassive StretchWall TensionActive ForcePotassium Chloride
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Konja, D., Luo, C., Sun, W. Y., Yang, K., Man, A. W., Xu, A., Vanhoutte, P. M., Wang, Y. Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues. J. Vis. Exp. (148), e59688, doi:10.3791/59688 (2019).
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