去细胞全喉软骨支架的免疫学研究

发布时间：2018-06-26 04:20

本文选题：喉 + 支架　；参考：《第四军医大学》2010年硕士论文

【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(P0.01)。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。
[Abstract]:The use of immune rejection and immune inhibitor is the main cause of successful laryngeal transplantation . The rapid and severe graft rejection reaction is the main obstacle to the success of clinical organ transplantation . In recent years , the study of cell scaffolds has provided hope for the construction of low immunogenicity throat . In recent years , the research of cell scaffolds has provided hope for the construction of low immunogenicity throat . However , the immunogenicity of the laryngeal scaffold treated by the decell technique is to be further confirmed . This study uses arterial perfusion to remove the muscle and mucous membrane of donor throat to retain its three - dimensional space structure , constructs the allograft artificial laryngeal cartilage scaffold and evaluates its immunogenicity , and provides the theoretical basis for the reconstruction of the whole throat .

Purpose

1 . To study the feasibility of constructing acellular cartilage scaffold by perfusion method , and to observe the morphological observation .

2 . To evaluate the immunogenicity of acellular laryngeal cartilage scaffold by animal experiment method , and provide theoretical basis for tissue engineering method to construct the whole - throat application .

method

1.To remove soluble matrix , the three - dimensional space structure of the extracellular matrix and the whole structure of whole laryngeal cartilage were retained .

2 . Immunologic study of acellular whole - throat scaffold

The experimental group : New Zealand white rabbits were used as the donor of the laryngeal scaffold , and the whole - throat stent of the muscle cells was constructed by the anterograde perfusion of the bilateral common carotid artery . The blue - violet blue rabbits were used as recipients , and the whole - throat stent was transplanted to the laryngeal muscles of the blue - violet rabbit . The control group : New Zealand rabbits were used as donors , and the isolated throat was directly implanted next to the laryngeal muscles of the blue - violet rabbit . All the groups were treated with 2 , 4 , 12 , 24 weeks after implantation .

Results

1 . The structure of acellular whole - throat stent and its morphological study were as follows : After 16 - hour perfusion of sodium dodecyl sulfate ( SDS ) , it was clear that the structure of the acellular laryngeal stent was intact and the color was pale . Scanning electron microscope ( SEM ) showed that the structure of the laryngeal muscle group was complete , the pores were much , the collagen fibers were not damaged , and the viability of the chondrocytes was intact .

2 . Immunologic study of acellular whole - throat scaffold

After 4 weeks , the cartilage structure disappeared and the cartilage structure disappeared and the connective tissue was replaced by connective tissue after 24 weeks . The lymphocyte infiltration in the experimental group was lower than that of the control group ( P0.01 ) .

Conclusion

1 . The perfusion method can eliminate the epithelial cells and muscle cells of the laryngeal tissue , retain the extracellular matrix and the cartilage , meet the requirement of the decell , and construct the complete acellular whole - throat bracket .

2 . The structure of the acellular cartilage scaffold is relatively intact , the immune rejection is mild , and the acellular cartilage scaffold has good biocompatibility , and is an alternative throat bracket material .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R767.9

【参考文献】