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Médecine

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Published: December 21, 2014
doi:
10.3791/52012
,
1Vascular Biology Program, Department of Surgery,Boston Children’s Hospital and Harvard Medical School

Summary

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Abstract

在干细胞研究，生物工程技术的最新显著的进步在利用生物材料的再生和修复，在骨科和牙周领域简单的组织损伤取得了很大进展。然而，试图再生的结构和更复杂的三维（3D）的器官的功能，如肺部并不是非常成功的，因为器官再生的生物过程还没有被很好的探讨。这是越来越明显，血管生成，新血管的形成，起着器官再生的关键角色。新形成的脉管不仅输送氧气，营养物质和所需要的器官再生的各种细胞成分，但也提供了有益的信号，以再生局部组织。因此，为了成功地再生成人在肺部，有必要概括的肺特异性的微环境，其中血管发生的局部肺组织的驱动器再生。虽然合作nventional 体内血管发生测定法，诸如细胞外基质（ECM）的富水凝胶（ 例如 ，纤维蛋白或胶原凝胶或基质胶-细胞外基质蛋白混合物通过Engelbreth-Holm的-群小鼠肉瘤细胞分泌）的皮下植入，被广泛用于探索血管生成的一般机制，肺特异性血管发生还没有得到很好的特点，因为在肺癌生物材料的原位移植方法尚未确立。该协议的目的是介绍一种独特的方法来植入活成年小鼠的肺表面纤维蛋白胶，允许主机肺源性血管生成的成功概括的凝胶内。这种方法使研究人员能够探索由肺特定的微环境控制血管形成和牙槽骨的再生在正常和病理条件下的机制。由于植入的生物材料释放并提供物理和化学信号到相邻的升UNG组织中，对患病的肺这些生物材料的植入可以潜在正常化相邻患病组织，使研究人员开发新的治疗方法用于各种类型的肺部疾病。

Introduction

该协议的总体目标是引进一种方法来对植入成年小鼠，这使研究人员能够表征肺血管和肺泡发育的分子机制，肺表面纤维蛋白胶，并为了发展仿生材料能够利用这些知识的扼要生理肺血管和肺泡的形成来治疗各种肺部疾病。

超过3500万美国人患有慢性肺部疾病，包括慢性阻塞性肺疾病，肺纤维化。这些患者有持久的慢性呼吸道症状，如呼吸急促，胸闷，咳嗽唠叨，和疲倦，其中显著影响他们的日常生活1-3。尽管努力开发有效的治疗这些肺病量很大，目前还没有治愈;因此，生活质量对这些患者差，经济和人力成本是喜GH 4-7。目前，肺移植是挽救患者的终末期慢性肺部疾病的唯一途径。由于移植的供体，成本高，严重的并发症，成活率低8-11短缺然而，移植不是一个最佳的方法。在组织工程技术的最新进展迅速使研究人员通过重新填充脱细胞的全肺与各种类型的祖细胞或诱导的多能干细胞（iPS细胞）12,13，以生物工程植入肺。然而，这些生物工程肺部功能的宿主动物只植入12,14,15后的几个小时。利用生物材料再生肺的复杂结构和功能也已经相当成功。这可能是因为支配成人肺再生键生物过程没有得到很好的研究。在肺，形成血管系统是国内最早，最重要的事件之一杜里吴发育和再生16-21。在肺中，不仅输送氧气，营养物质和所需的器官形成的各种细胞成分，而且还提供了有益的调节信号到周围的细胞22-25新形成的脉管。因此，血管生成在成年肺部24,26,27再生alveolarization关键作用。此外，失调的血管生成有助于慢性肺部疾病，如慢性阻塞性肺疾病（COPD）28，支气管肺发育不良（BPD）21-23，和肺纤维化29。因此，开发用于工程肺或治疗慢性肺疾病更有效的策略，有必要了解肺特异性血管生成的基本机制。

每个器官显示独特的机械和化学性质，其可生理和病理条件30-33之间不同。这些器官特异性microenvironments调节内皮细胞行为和协调在器官特异性方式24,34-36血管网形成。因此，开发用于肺再生更有效的策略，需要底层肺特异性血管生成的机制，来理解。而常规的体内血管发生测定法，如皮下植入的水凝胶已被广泛地用于研究血管发生37-39，这些方法不重述器官特异性血管生成。最近，一种新的方法，以在植入的小鼠的肺的弹性模基底膜已经开发并示出成功地招募血管和肺上皮细胞进入凝胶22。这种独特的方法将允许研究人员探索生理和病理条件下肺特异性血管生成的血管和非血管肺细胞的机制，以及相互作用。自1）的Matrigel不适合临床应用; 2）电子商务用于铸造的凝胶可能影响水凝胶和宿主肺组织和3）对肺的弹性模呼吸期间潜在地导致肺功能和疼痛的障碍之间的相互作用，作为更临床相关的方式LASTIC模​​具，三维纤维蛋白基质含有血管生成因子（血管内皮生长因子（VEGF）/碱性成纤维细胞生长因子（bFGF））已被植入在小鼠肺而不浇铸在弹性模，并已成功地概括宿主肺源性血管生成。纤维蛋白胶，从凝血酶裂解纤维蛋白原产生聚合物纤维，是已知的陷阱多种血管生成因子，如bFGF和VEGF的加速血管生成在体内 40,41。因为它的再生能力和可生物降解的性质的42，纤维蛋白胶被广泛应用于组织工程领域。

本文介绍了一种新颖而独特的方式植入活adul肺表面纤维蛋白胶吨小鼠并表明宿主肺源性血管生成被概括在体内的凝胶内。该方法中，这使得研究人员研究肺特异性血管生成，将有可能导致新的治疗方法用于各种类型的肺部疾病的发展和显著前进努力成功再生成人肺。

Protocol

注： 在动物体内进行了研究，在严格按照指南中的美国国立卫生研究院的实验动物的护理和使用的建议。该协议是审查和批准由波士顿儿童医院的动物护理和使用委员会（协议编号：13-10-2526R，14-02-2568R）。在这个协议中使用的所有药品都是医药级和这些药物在无菌条件下制备。 1.纤维蛋白胶的制备准备包含VEGF和bFGF纤维蛋白胶。 纤维蛋白原和凝血…

Representative Results

检查宿主肺源性血管形成是否被概括植入肺中的生物材料的内部，纤维蛋白凝胶补充有大血管生成因子VEGF和bFGF（0,10和100ng / ml的各自）植入的活小鼠肺部的表面上使用基底膜22日报道。纤维蛋白凝胶47包含这些血管生成生长因子被制造， 如图1a所示 。开胸后，左肺表面的一个小区域，使用镊子刮下和所制造的纤维蛋白凝胶植入在成年小鼠的使用量小的纤维蛋白胶的肺?…

Discussion

本文介绍了一种新的方法来植入活的成年小鼠的肺表面的生物材料。有了这个系统，主机肺源性血管被成功概括材料内部。这个系统使研究人员能够内皮细胞之间探索串扰，其他细胞（ 例如 ，上皮细胞，间质细胞，免疫细胞）和各种细胞外基质成分所需要的地方的血管生成50-53和肺泡再生24,54。尽管传统的体内皮下植入的水凝胶已被广泛地用于研究血管发生37-39?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国心脏协会（AM），美国国防部（BC074986）和波士顿儿童医院学院职业发展奖学金（TM，AM）的资金支持。作者感谢阿曼达江江伊丽莎白的技术援助。

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Fibrinogen from human placenta Sigma F4883 For fabrication of fibrin gel
Thrombin from bovine plasma Sigma T9549 For fabrication of fibrin gel
Recombinant mouse VEGF 164 R&D 493-MV For supplementation to fibrin gel
Recombinant mouse bFGF R&D 3139-FB For supplementation to fibrin gel
Rodent Intubation Stand Braintree Scientific INC RIS 100 For intubation
Fiber-Optic Light Source Fisher Scientific 12-565-35 For intubation
20G Elastic catheter B.Braun 4251652-02 For intubation
MiniVent Ventilator Harvard Apparatus CGS-8009 For ventilation
Stemi DV4 Steromicroscope Fisher Scientific 12-070-515 For surgey
Absobable suture Ethicon PDP304 Surgical suture
Antibody against CD31 BD Biosciences 553370 Immunohistochemistry
Antibody against AQP5 Abcam AB78486 Immunohistochemistry
Antibody against SP-B Millipore AB40876 Immunohistochemistry
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Tags

Keywords: AngiogenesisFibrin GelLung RegenerationBiomaterialsImplantationOrthotopicMouse ModelLung-specific MicroenvironmentStem Cell Research
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Mammoto, T., Mammoto, A. Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis. J. Vis. Exp. (94), e52012, doi:10.3791/52012 (2014).
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