本文選題：喉 + 支架��； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 喉移植是全喉切除后恢復(fù)喉功能的理想方法,而免疫排斥反應(yīng)和免疫抑制劑的使用是限制喉移植開展的主要原因�？焖俣鴩�(yán)重的移植排斥反應(yīng)是臨床器官移植成功的主要障礙。如能構(gòu)建低免疫原性生物人工喉則可望解決這一問題。近年來去細(xì)胞支架的研究為構(gòu)建低免疫原性喉提供了希望。去細(xì)胞生物支架是用物理或化學(xué)等方法去除組織或器官內(nèi)的細(xì)胞和可溶性蛋白,僅保留組織結(jié)構(gòu)中的不溶性基質(zhì)成分,其主要成份為膠原,有助于種子細(xì)胞的粘附,增值,生長,分化,可為新組織的生成提供網(wǎng)狀結(jié)構(gòu)支架[1]。但這種經(jīng)去細(xì)胞技術(shù)處理的喉支架的免疫原性有待進一步證實。本研究用動脈灌注去細(xì)胞技術(shù)去除供體喉的肌肉和黏膜而保留其三維空間結(jié)構(gòu),構(gòu)建同種異體生物人工喉軟骨支架并評價其免疫原性,為全喉重建提供理論依據(jù)。 目的 1.通過灌注法構(gòu)建去細(xì)胞全喉支架,探討灌注法構(gòu)建去細(xì)胞軟骨支架的可行性,并對其進行形態(tài)學(xué)觀察。 2.通過動物體內(nèi)實驗法評價去細(xì)胞喉軟骨支架的免疫原性,為組織工程方法構(gòu)建全喉的應(yīng)用提供理論依據(jù)。 方法 1.去細(xì)胞全喉支架的構(gòu)建及其形態(tài)學(xué)研究通過頸總動脈灌注離子型去污劑,對離體全喉進行去細(xì)胞處理。在去除可溶性基質(zhì)的同時保留喉細(xì)胞外基質(zhì)的三維空間結(jié)構(gòu)及完整的全喉軟骨支架結(jié)構(gòu),制備后的去細(xì)胞喉支架行大體觀察、掃描電鏡觀察及組織學(xué)觀察。 2.去細(xì)胞全喉支架的免疫學(xué)研究 實驗組:新西蘭大白兔作為喉支架供體,通過雙側(cè)頸總動脈順行灌注去離子劑,構(gòu)建出去肌細(xì)胞全喉支架。青紫藍兔為受體,將去細(xì)胞全喉支架植入青紫蘭兔喉肌旁;對照組:新西蘭大白兔新鮮離體喉作供體,青紫蘭兔為受體,將離體喉直接植入青紫蘭兔喉肌旁,各組分別于植入后2,4,12,24周取材進行大體觀察、組織學(xué)觀察及淋巴細(xì)胞浸潤測定。 結(jié)果 1.去細(xì)胞全喉支架的構(gòu)建及其形態(tài)學(xué)研究大體觀察:離體喉經(jīng)十二烷基硫酸鈉(SDS)灌注十六小時后即透明,去細(xì)胞喉支架結(jié)構(gòu)完整,顏色蒼白。掃描電鏡觀察:灌注組喉肌群去細(xì)胞程度完全,孔隙多,膠原纖維未受損,且軟骨細(xì)胞活力保存完好。去細(xì)胞喉冠狀切面HE染色示:喉聲帶、室?guī)А椥詧A錐等結(jié)構(gòu)均存在,肌細(xì)胞完全被去除,纖維樣結(jié)構(gòu)仍存在,排列規(guī)整,較完整的保留了細(xì)胞外基質(zhì)的方向性。 2.去細(xì)胞全喉支架的免疫學(xué)研究 各組動物術(shù)后均存活至取材時間,實驗組傷口愈合較對照組好。組織學(xué)觀察:對照組2周出現(xiàn)免疫排斥反應(yīng),4周后軟骨結(jié)構(gòu)被破壞,喉的基本結(jié)構(gòu)消失;實驗組2周時出現(xiàn)輕微的炎性反應(yīng),4周時形成纖維結(jié)締組織包繞,炎性反應(yīng)減輕,12周軟骨結(jié)構(gòu)存在,軟骨周圍大量結(jié)締組織形成,24周后,軟骨結(jié)構(gòu)消失,被結(jié)締組織取代。淋巴細(xì)胞浸潤觀察:各時間點實驗組淋巴細(xì)胞浸潤程度均低于對照組(P0.01)。 結(jié)論 1.灌注法能夠清除喉組織的上皮細(xì)胞及肌細(xì)胞,保留細(xì)胞外基質(zhì)及軟骨,達到去細(xì)胞要求,構(gòu)建完整的去細(xì)胞全喉支架。 2.去細(xì)胞軟骨支架結(jié)構(gòu)相對完整,免疫排斥輕微,具有良好的生物相容性,是一種可供選擇的喉支架材料。
[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 .
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R767.9

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