去细胞全喉软骨支架的免疫学研究
摘要
喉移植是全喉切除后恢复喉功能的理想方法,而免疫排斥反应和免疫抑制剂的使用是限制喉移植开展的主要原因。快速而严重的移植排斥反应是临床器官移植成功的主要障碍。如能构建低免疫原性生物人工喉则可望解决这一问题。近年来去细胞支架的研究为构建低免疫原性喉提供了希望。去细胞生物支架是用物理或化学等方法去除组织或器官内的细胞和可溶性蛋白,仅保留组织结构中的不溶性基质成分,其主要成份为胶原,有助于种子细胞的粘附,增值,生长,分化,可为新组织的生成提供网状结构支架[1]。但这种经去细胞技术处理的喉支架的免疫原性有待进一步证实。本研究用动脉灌注去细胞技术去除供体喉的肌肉和黏膜而保留其三维空间结构,构建同种异体生物人工喉软骨支架并评价其免疫原性,为全喉重建提供理论依据。
目的
1.通过灌注法构建去细胞全喉支架,探讨灌注法构建去细胞软骨支架的可行性,并对其进行形态学观察。
2.通过动物体内实验法评价去细胞喉软骨支架的免疫原性,为组织工程方法构建全喉的应用提供理论依据。
方法
1.去细胞全喉支架的构建及其形态学研究通过颈总动脉灌注离子型去污剂,对离体全喉进行去细胞处理。在去除可溶性基质的同时保留喉细胞外基质的三维空间结构及完整的全喉软骨支架结构,制备后的去细胞喉支架行大体观察、扫描电镜观察及组织学观察。
2.去细胞全喉支架的免疫学研究
实验组:新西兰大白兔作为喉支架供体,通过双侧颈总动脉顺行灌注去离子剂,构建出去肌细胞全喉支架。青紫蓝兔为受体,将去细胞全喉支架植入青紫兰兔喉肌旁;对照组:新西兰大白兔新鲜离体喉作供体,青紫兰兔为受体,将离体喉直接植入青紫兰兔喉肌旁,各组分别于植入后2,4,12,24周取材进行大体观察、组织学观察及淋巴细胞浸润测定。
结果
1.去细胞全喉支架的构建及其形态学研究大体观察:离体喉经十二烷基硫酸钠(SDS)灌注十六小时后即透明,去细胞喉支架结构完整,颜色苍白。扫描电镜观察:灌注组喉肌群去细胞程度完全,孔隙多,胶原纤维未受损,且软骨细胞活力保存完好。去细胞喉冠状切面HE染色示:喉声带、室带、弹性圆锥等结构均存在,肌细胞完全被去除,纤维样结构仍存在,排列规整,较完整的保留了细胞外基质的方向性。
2.去细胞全喉支架的免疫学研究
各组动物术后均存活至取材时间,实验组伤口愈合较对照组好。组织学观察:对照组2周出现免疫排斥反应,4周后软骨结构被破坏,喉的基本结构消失;实验组2周时出现轻微的炎性反应,4周时形成纤维结缔组织包绕,炎性反应减轻,12周软骨结构存在,软骨周围大量结缔组织形成,24周后,软骨结构消失,被结缔组织取代。淋巴细胞浸润观察:各时间点实验组淋巴细胞浸润程度均低于对照组(P<0.01)。
结论
1.灌注法能够清除喉组织的上皮细胞及肌细胞,保留细胞外基质及软骨,达到去细胞要求,构建完整的去细胞全喉支架。
2.去细胞软骨支架结构相对完整,免疫排斥轻微,具有良好的生物相容性,是一种可供选择的喉支架材料。
Laryngeal transplantation is an ideal way to restore laryngeal function aftertotal laryngectomy. The first real problem faced by laryngeal transplantation isthe immune rejection and the use of immunosuppressive agent. Rapid andsevere immune rejection is a major obstacle to successful transplantation. If wecan construct a low immunogenicity bio-artificial larynx ,this problem may besolved. Rencently ,with the rapid development of tissue engineering , acellularscaffold brings hope to construct a low immunity larynx . Acellular scaffold isobtained by chemical detergent, which removed cells and soluble protein of thetissues or organs, retaining only the organizational structure of the insolublematrix components. The extracellular matrixs (ECMs) mainly contain collagen.The collagen provide scaffolds for cell adherence, cell migration,proliferationand tissue growth, . However, there is no research about the immunogenicity ofthe decellularized laryngeal scaffold. In our study, we attempted to construct adecellularized laryngeal collagen scaffold which reserved three-dimensionalgeometry extracellular matrix by utilizing a perfusion-decellularized technique and evaluate the immunogenicity of the decellularized laryngeal scaffold .
Objectives
1. To prepare an extracellular matrix laryngeal scaffold by utilizing a perfused,decellularized technique and investigate its feasibility to be athree-dimensional geometry extracellular matrix whole laryngeal collagenscaffold. Morphological observation was conducted on the decellularizedwhole laryngeal scaffold.
2. To appraise the decellularized whole laryngeal scaffold’s immunogenicity byimplanted the scaffold in para-laryngeal muscles in rabbits, which will set atheoretical foundation for the application in the tissue engineering.
Methods
1. Morphological observation on the decellularized whole laryngeal collagenscaffold
Perfusion decelluarized whole laryngeal collagen scaffolds were obtainedby carotid arterious perfusion with detergents. They were evaluated bymacroscopic view, scanning electron microscope (SEM) and histologicalexamination.
2. Immunological study on decellularized whole laryngeal scaffold
Twelve perfusion decelluarized laryngeal scaffolds were obtained fromrabbits through perfusion detergents by common carotid arterious. The twelvedecellularized laryngeal scaffolds and the twelve fresh larynxes were separatelyimplanted in para-laryngeal muscles of rabbits and harvested after two weeks,four weeks, twelve weeks and twenty-four weeks, respectively. Then, performedmacroscopic view, histological examination and lymphocyte infiltration test onthese samples.
Results
1. Morphological observation on the decellularized whole laryngeal scaffold
Macroscopic view showed that the decellularized whole laryngeal collagenscaffold became transparent after two hours perfusion by detergents SDS.Perfusion decellularized laryngeal collagen scaffold can preserve the laryngealextracellular matrix and produce an acellular whole laryngeal collagenarchitecture. Histology and SEM indicated that the perfused laryngeal scaffoldhad a better deculluarized effect. The ventages and collagen fibers were retainedperfectly. HE staining of the coronal section of decellularized larynx indicatedthat the structure of laryngeal vocal cord, ventricular band, elastic cone wasintact. Muscle cells was completely removed.The fiber-like structure and thedirection of the extracellular matrix were remained.
2. Immunological study on decellularized whole laryngeal scaffold
The decelluarized larynx did not show obvious immunological rejectionafter implanted in the para-laryngeal muscles of the reeipient rabbits. Thevolume of implanted larynx became smaller but retain cartilage scaffold.Larynxes in the control group presented the serious immunological rejection andthe majority tissues of the larynxes were disintegrated and substituted by thefibrous connective tissues after four weeks. The peripheral tissues weredamaged and necrotic at different degrees. The quantity of the lymphocyteinfiltration in the control group is higher than that in the experiment group andthe result had the statistical significance (P<0.01).
Conclusions
1. Perfused, decellularized technique can achieve deculluarized effect. Theepithelial cells and muscle cells were removed, while extracellular matrix and cartilage were retained. The perfused technique can preserve thelaryngeal extracellular matrix and cartilage framework and construct anacellular whole laryngeal collagen scaffold.
2. Decellularized larynx scaffold shows a low immunity laryngeal which couldbe a satisfactory material for laryngeal repair.
目的
1.通过灌注法构建去细胞全喉支架,探讨灌注法构建去细胞软骨支架的可行性,并对其进行形态学观察。
2.通过动物体内实验法评价去细胞喉软骨支架的免疫原性,为组织工程方法构建全喉的应用提供理论依据。
方法
1.去细胞全喉支架的构建及其形态学研究通过颈总动脉灌注离子型去污剂,对离体全喉进行去细胞处理。在去除可溶性基质的同时保留喉细胞外基质的三维空间结构及完整的全喉软骨支架结构,制备后的去细胞喉支架行大体观察、扫描电镜观察及组织学观察。
2.去细胞全喉支架的免疫学研究
实验组:新西兰大白兔作为喉支架供体,通过双侧颈总动脉顺行灌注去离子剂,构建出去肌细胞全喉支架。青紫蓝兔为受体,将去细胞全喉支架植入青紫兰兔喉肌旁;对照组:新西兰大白兔新鲜离体喉作供体,青紫兰兔为受体,将离体喉直接植入青紫兰兔喉肌旁,各组分别于植入后2,4,12,24周取材进行大体观察、组织学观察及淋巴细胞浸润测定。
结果
1.去细胞全喉支架的构建及其形态学研究大体观察:离体喉经十二烷基硫酸钠(SDS)灌注十六小时后即透明,去细胞喉支架结构完整,颜色苍白。扫描电镜观察:灌注组喉肌群去细胞程度完全,孔隙多,胶原纤维未受损,且软骨细胞活力保存完好。去细胞喉冠状切面HE染色示:喉声带、室带、弹性圆锥等结构均存在,肌细胞完全被去除,纤维样结构仍存在,排列规整,较完整的保留了细胞外基质的方向性。
2.去细胞全喉支架的免疫学研究
各组动物术后均存活至取材时间,实验组伤口愈合较对照组好。组织学观察:对照组2周出现免疫排斥反应,4周后软骨结构被破坏,喉的基本结构消失;实验组2周时出现轻微的炎性反应,4周时形成纤维结缔组织包绕,炎性反应减轻,12周软骨结构存在,软骨周围大量结缔组织形成,24周后,软骨结构消失,被结缔组织取代。淋巴细胞浸润观察:各时间点实验组淋巴细胞浸润程度均低于对照组(P<0.01)。
结论
1.灌注法能够清除喉组织的上皮细胞及肌细胞,保留细胞外基质及软骨,达到去细胞要求,构建完整的去细胞全喉支架。
2.去细胞软骨支架结构相对完整,免疫排斥轻微,具有良好的生物相容性,是一种可供选择的喉支架材料。
Laryngeal transplantation is an ideal way to restore laryngeal function aftertotal laryngectomy. The first real problem faced by laryngeal transplantation isthe immune rejection and the use of immunosuppressive agent. Rapid andsevere immune rejection is a major obstacle to successful transplantation. If wecan construct a low immunogenicity bio-artificial larynx ,this problem may besolved. Rencently ,with the rapid development of tissue engineering , acellularscaffold brings hope to construct a low immunity larynx . Acellular scaffold isobtained by chemical detergent, which removed cells and soluble protein of thetissues or organs, retaining only the organizational structure of the insolublematrix components. The extracellular matrixs (ECMs) mainly contain collagen.The collagen provide scaffolds for cell adherence, cell migration,proliferationand tissue growth, . However, there is no research about the immunogenicity ofthe decellularized laryngeal scaffold. In our study, we attempted to construct adecellularized laryngeal collagen scaffold which reserved three-dimensionalgeometry extracellular matrix by utilizing a perfusion-decellularized technique and evaluate the immunogenicity of the decellularized laryngeal scaffold .
Objectives
1. To prepare an extracellular matrix laryngeal scaffold by utilizing a perfused,decellularized technique and investigate its feasibility to be athree-dimensional geometry extracellular matrix whole laryngeal collagenscaffold. Morphological observation was conducted on the decellularizedwhole laryngeal scaffold.
2. To appraise the decellularized whole laryngeal scaffold’s immunogenicity byimplanted the scaffold in para-laryngeal muscles in rabbits, which will set atheoretical foundation for the application in the tissue engineering.
Methods
1. Morphological observation on the decellularized whole laryngeal collagenscaffold
Perfusion decelluarized whole laryngeal collagen scaffolds were obtainedby carotid arterious perfusion with detergents. They were evaluated bymacroscopic view, scanning electron microscope (SEM) and histologicalexamination.
2. Immunological study on decellularized whole laryngeal scaffold
Twelve perfusion decelluarized laryngeal scaffolds were obtained fromrabbits through perfusion detergents by common carotid arterious. The twelvedecellularized laryngeal scaffolds and the twelve fresh larynxes were separatelyimplanted in para-laryngeal muscles of rabbits and harvested after two weeks,four weeks, twelve weeks and twenty-four weeks, respectively. Then, performedmacroscopic view, histological examination and lymphocyte infiltration test onthese samples.
Results
1. Morphological observation on the decellularized whole laryngeal scaffold
Macroscopic view showed that the decellularized whole laryngeal collagenscaffold became transparent after two hours perfusion by detergents SDS.Perfusion decellularized laryngeal collagen scaffold can preserve the laryngealextracellular matrix and produce an acellular whole laryngeal collagenarchitecture. Histology and SEM indicated that the perfused laryngeal scaffoldhad a better deculluarized effect. The ventages and collagen fibers were retainedperfectly. HE staining of the coronal section of decellularized larynx indicatedthat the structure of laryngeal vocal cord, ventricular band, elastic cone wasintact. Muscle cells was completely removed.The fiber-like structure and thedirection of the extracellular matrix were remained.
2. Immunological study on decellularized whole laryngeal scaffold
The decelluarized larynx did not show obvious immunological rejectionafter implanted in the para-laryngeal muscles of the reeipient rabbits. Thevolume of implanted larynx became smaller but retain cartilage scaffold.Larynxes in the control group presented the serious immunological rejection andthe majority tissues of the larynxes were disintegrated and substituted by thefibrous connective tissues after four weeks. The peripheral tissues weredamaged and necrotic at different degrees. The quantity of the lymphocyteinfiltration in the control group is higher than that in the experiment group andthe result had the statistical significance (P<0.01).
Conclusions
1. Perfused, decellularized technique can achieve deculluarized effect. Theepithelial cells and muscle cells were removed, while extracellular matrix and cartilage were retained. The perfused technique can preserve thelaryngeal extracellular matrix and cartilage framework and construct anacellular whole laryngeal collagen scaffold.
2. Decellularized larynx scaffold shows a low immunity laryngeal which couldbe a satisfactory material for laryngeal repair.
引文
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