da Palma Renata Kelly, Fratini Paula, Schiavo Matias Gustavo Sá, Cereta Andressa Daronco, Guimarães Leticia Lopes, Anunciação Adriana Raquel de Almeida, de Oliveira Luis Vicente Franco, Farre Ramon, Miglino Maria Angelica
Post Graduate Program in Science of Rehabilitation, University Nove de Julho (UNINOVE), São Paulo, Brazil.
Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
J Tissue Eng. 2018 Nov 12;9:2041731418810164. doi: 10.1177/2041731418810164. eCollection 2018 Jan-Dec.
Contrary to conventional research animals, horses naturally develop asthma, a disease in which the extracellular matrix of the lung plays a significant role. Hence, the horse lung extracellular matrix appears to be an ideal candidate model for in vitro studying the mechanisms and potential treatments for asthma. However, so far, such model to study cell-extracellular matrix interactions in asthma has not been developed. The aim of this study was to establish a protocol for equine lung decellularization that maintains the architecture of the extracellular matrix and could be used in the future as an in vitro model for therapeutic treatment in asthma. For this the equine lungs were decellularized by sodium dodecyl sulfate detergent perfusion at constant gravitational pressure of 30 cmHO. Lung scaffolds were assessed by immunohistochemistry (collagen I, III, IV, laminin, and fibronectin), scanning electron microscopy, and DNA quantification. Their mechanical property was assessed by measuring lung compliance using the super-syringe technique. The optimized protocol of lung equine decellularization was effective to remove cells (19.8 ng/mg) and to preserve collagen I, III, IV, laminin, and fibronectin. Moreover, scanning electron microscopy analysis demonstrated maintained microscopic lung structures. The decellularized lungs presented lower compliance compared to native lung. In conclusion we described a reproducible decellularization protocol that can produce an acellular equine lung feasible for the future development of novel treatment strategies in asthma.
与传统实验动物不同,马会自然患上哮喘,在这种疾病中,肺的细胞外基质起着重要作用。因此,马肺细胞外基质似乎是体外研究哮喘发病机制和潜在治疗方法的理想候选模型。然而,到目前为止,尚未开发出用于研究哮喘中细胞与细胞外基质相互作用的此类模型。本研究的目的是建立一种马肺脱细胞方案,该方案能维持细胞外基质的结构,并可在未来用作哮喘治疗的体外模型。为此,通过在30 cmHO的恒定重力压力下用十二烷基硫酸钠洗涤剂灌注使马肺脱细胞。通过免疫组织化学(胶原蛋白I、III、IV、层粘连蛋白和纤连蛋白)、扫描电子显微镜和DNA定量对肺支架进行评估。通过使用超级注射器技术测量肺顺应性来评估其力学性能。优化后的马肺脱细胞方案能有效去除细胞(19.8 ng/mg)并保留胶原蛋白I、III、IV、层粘连蛋白和纤连蛋白。此外,扫描电子显微镜分析显示肺微观结构得以保留。与天然肺相比,脱细胞肺的顺应性较低。总之,我们描述了一种可重复的脱细胞方案,该方案能够产生一种无细胞的马肺,这对于未来哮喘新治疗策略的开发是可行的。