【作者】 陈昶；

【导师】 沈振亚；

【作者基本信息】 苏州大学 ， 胸心血管外科， 2009， 博士

【摘要】 研究背景:“再上皮化”是同种异体原位气管移植所面临的最大难题之一,目前仍然停留在实验阶段。同种异体原位气管移植后的再上皮化过程一旦不能完成,则移植气管将受到免疫排斥因而发生粘膜下结缔组织增生、淋巴细胞浸润、气管管腔闭塞等一系列的病理改变,即称为OAD(气道闭塞性疾病,Obliterative Airway Disease)。关于上皮组织再生对于OAD发生的影响,实验研究集中采用啮齿类动物的异位气管移植和原位气管移植两种模型得以实现。本实验拟针对上述两种模型的建立方法和OAD病理组织学特征、该模型中受体上皮再生对于防止OAD的作用、以及在此模型中应用细胞种植的方法实现上皮再生、维护上皮完整性、改善和防止OAD发生等方面进行研究。第一部分代表OAD典型病理结果的鼠气管移植模型。研究目的:建立代表OAD典型病理结果的鼠异位和原位气管移植模型。材料和方法:SPF级BALB/c(供体)和C57BL/6(受体)小鼠共80只,分别设立实验组(异系移植)和对照组(同系移植),并分别建立原位和异位气管移植。获取供体小鼠的长段气管后,用手术方法直接种植于同种异系的受体鼠背部皮下,建立异位气管移植模型。另取供体小鼠的长段气管,用手术方法与受体鼠的气管行端端吻合,建立原位移植模型。结果:异系小鼠行异位移植四周后,供体气管段发生显著的纤维结缔组织增生、大量淋巴细胞浸润、管腔闭塞等,为典型的OAD病理表现。原位气管移植4周后该移植气管管腔通畅,纤毛上皮完整,粘膜下组织轻度增生,少量淋巴细胞浸润,和异位移植模型构成了显著的病理组织学对比。结论:通过本实验建立了具有典型OAD病理表现的实验动物模型。原位气管移植和异位气管移植的不同病理结局,说明两种移植方式后的供体气管发生了组织学上的重新构建。第二部分受体来源的上皮细胞再生对于OAD发生的作用。研究目的:利用鼠异位和原位气管移植模型,证明受体来源的上皮细胞再生是防止OAD发生的关键环节。材料和方法:SPF级BALB/c(供体)和C57BL/6(受体)小鼠共80只,分别设为四组:异位异系、原位异系、异位同系、原位同系组。移植后4周,分别观察移植气管段的病理组织学特征、作定量形态学分析(LCR、粘膜下层纤维组织的面积、纤毛上皮的比例)、检测血CD4+CD8+淋巴细胞表达率、并鉴定移植气管的上皮表型。结果:①所有供受体同为Balb/c的实验组,测定的LCR、纤维组织面积和纤毛上皮比例和正常气管相比,统计学无明显差异。异位异系小鼠间的气管移植(Balb/c→B1/6),由于在4周终点所有移植气管的管腔均发生闭塞,只能做纤维组织面积的粗略测定,与正常气管相比P＜0.01,与其它各组相比P＜0.05。所有原位异系小鼠的气管移植,即(Balb/c→B1/6,颈部原位移植),测得的LCR和正常气管统计学差异显著P＜0.05,纤毛上皮比例亦有统计学差异P＜0.05。原位同系小鼠间的移植气管,在LCR、纤维像素面积、纤毛上皮比率等指标上,与正常气管无统计学差异。②原位移植的供体Balb/c气管,具有阳性的B1/6小鼠的上皮抗原性表达(H2Kd抗原)。③各实验组的小鼠CD4+/CD8+阳性表达率的统计学差异不明显(P＞0.05)。结论:在原位气管移植模型中,由于受体上皮细胞能够移行和充分覆盖供体气管并完成再上皮化,从而能维持供体气管的管腔通畅。气管上皮组织的抗原性来源是OAD发生的关键环节。第三部分上皮细胞种植对于防止OAD发生的作用。研究目的:探讨受体来源的上皮细胞种植对于防止供体裸气管发生OAD病理结局的作用。材料和方法:①SPF级SD大鼠(受体)和Wistar大鼠(供体)各30只;②SD大鼠上皮细胞行体外培养;③所有移植气管获取后,进行酶消化上皮细胞,制成供体裸气管;④实验设为四组,分别取体外培养的SD大鼠上皮细胞、消化得到的Wistar大鼠上皮细胞、培养基和0.9%氯化钠注射液进行供体Wistar大鼠裸气管的管腔内灌注,然后行异位气管移植,将供体气管种植于受体SD大鼠皮下。移植气管于移植后4周获取,行病理组织学检查。同时检测受体鼠的血CD4+/CD8+T淋巴细胞比率。结果:上皮细胞灌注的实验组气管,管腔维持通畅完整,粘膜下结缔组织有部分增生,管腔内可见到较为完整的纤毛上皮组织。培养基和生理盐水灌注组的病理组织学特征相类似,均表现为管腔的闭锁、上皮组织缺失、显著的淋巴细胞浸润和管腔内的纤维结缔组织增生,即典型的OAD特征。受体大鼠的CD4+/CD8+比率,上皮细胞种植组为1.5±0.3,和生理盐水灌注的对照组1.6±0.3无明显统计学差异(P＞0.05)。结论:上皮组织的抗原性和完整性都是影响同种异体气管移植中OAD病理结果的必要因素。外源性上皮细胞在管腔内的存在、并形成具有受体抗原性(SD大鼠)的上皮组织层,有效的防止了OAD的发生。提示体外培养的上皮细胞可能通过种植的方式,达到完全的再上皮化。第四部分细胞种植后新生上皮组织的完整性和屏障功能。研究目的:探讨受体来源的上皮细胞种植于供体气管管腔后,是否能建立屏障功能并维护上皮组织的完整性。材料和方法:①SPF级SD大鼠(受体)和Wistar大鼠(供体)各30只:②SD大鼠上皮细胞行体外培养;③所有移植气管获取后,进行酶消化上皮细胞,制成供体裸气管;④实验设为三组,分别取体外培养的SD大鼠上皮细胞、培养基、和0.9%氯化钠注射液进行供体Wistar大鼠裸气管的管腔内灌注,然后行异位气管移植,将供体Wistar大鼠气管种植于受体SD大鼠皮下。移植气管于移植后4周获取,行病理组织学、上皮组织的超微结构检查;行移植气管的CK14、CK18、CFTR和ZO-1免疫组化染色。结果:①新形成的柱状上皮细胞具有明显的纤毛结构,具有完整的细胞器结构,含有丰富的线粒体。上皮细胞间距离窄,且己具备完整的紧密连接。②新生的上皮组织层中,CK14、ZO-1和CFTR呈阳性表达。结论:体外培养的上皮细胞可以通过种植的手段,在异系受体鼠体内形成具有较为完整的屏障功能的上皮结构,从而得到保护供体气管、维持管腔通畅的病理结局。总结:①鼠原位和异位气管移植模型反映了OAD的病理结果,可作为OAD研究的动物模型;②鼠原位和异位气管移植得到的迥然相异的OAD病理表现,充分提示了气管上皮组织在OAD发生发展中的重要作用;③上皮组织的表型转换(再上皮化,即供体气管段具有受体源性的上皮组织),是维持供体气管通畅的关键环节;④通过体外培养气道上皮细胞,再进行供体裸气管管腔内的种植,将能够达到保护供体气管、维持管腔通畅的作用;⑤向供体气管管腔内种植的上皮细胞,能有效分化为较成熟的上皮组织,并具有较为完善的屏障功能。更多还原

【Abstract】 Background and PurposeRe-epithelialization remains one of the major obstacles for allogenic orthotopic tracheal transplantation,and is still under laboratory research.The transplanted tracheal allograft will undergo a series of pathological changes if the process of re-epithelialization has not been fulfilled.These pathological changes are named as Obliterative Airway Disease(OAD),which consist of submucous fibroproliferation,lymph infiltration and luminal obliteration.The two rodent tracheal transplantation models,orthotopic and heterotopic tracheal transplantations,are commonly adopted for the study of re-epithelialization and OAD.The Ppresent study aims to establish the experimental models of OAD,and characterize the histo-pathological manifestations of OAD,the role of re-epithelialization in OAD development,and the methods of epithelial implantation for epithelial repopulation.Chapter One The rodent tracheal transplantation models were established as representative of OAD pathological characteristics.Objective:To establish the rodent orthotopic and heterotopic tracheal transplantation models representative of OAD histo-pathology.Materials and Methods: A total of 80 Balb/c(donor) and C57BL/6 mice were included for the experiments of orthotopic and heterotopic tracheal transplantations.Heterotopic tracheal transplantation was accomplished by surgical implantation of donor allo-tracheal segments into the back pouch of the recipients,while orthotopic tracheal transplantation was prepared by end-to-end anastmosis to donor trachea.Results:The pathological appearances of the allografts at 4 weeks after heterotopic transplantations included prominent submucous fibroproliferation,lympho-infiltration and luminal obliteration which were consistent with distinctive OAD manifestations.The pathological manifestations of orthotopic transplantations included patent lumen of the allograft,integrated airway epithelium,mild submucous fibroproliferation,and little lympho-infiltration.These histo-pathological manifestations were obviously different between these two tracheal transplantation models. Conclusions:The present study has established experimental OAD models in rodents. The discrepancies between these two models suggest different underlying tissue re-modulations.Chapter Two The role of the recipient driving epithelial cells repopulation in OAD development was examined.Objective:To study the role of the recipient driving epithelial cells repopulation in OAD development.Materials and Methods:A total of 80 Balb/c(donor) and C57BL/6 mice were included for the experiments of orthotopic and heterotopic tracheal transplantations.There were four experimental groups,including orthotopic,hetertopic, allogenic and isogenic transplantations in which the histo-pathological examination, quantitative morphologic measurement(including LCR,submucous fibroproliferation and ratio of ciliated epithelium),CD4+/CD8+ ratio measurement,and epithelial phenotype stain were performed.Results:No OAD manifestations were noted in isogenic transplantations.There were significant differences between the heterotopically transplanted tracheas and the normal ones.H2Kd stain was positive in orthotopical Balb/c allografts.There were no differences amongst all four groups.Conclusions:Process of re-epithelialization was fulfilled in orthotopic allo-transplantations,due to the migration and re-population of the recipient epithelial cells.Such process contributed to the maintenance of a patent lumen.The antigenic expression of donor epithelium plays a key role in OAD development. Chapter Three The role of epithelial implantation in OAD development was examined.Objective:To study the role of recipient epithelial implantation in preventing OAD development.Materials and Methods:There were 60 SD rats and Wistar rats included in this experiment.After ex-vivo culture,the SD rat-origin epithelial cells were inoculated into the tracheal segments of nude Wistar rat,which were then heterotopically transplanted into recipient SD rats.The allografts underwent histopathological examinations four weeks after transplantation.The CD4+/CD8+ ratio of recipient rats was, as well,measured and compared amongst groups.Results:The allografts with epithelial implantation maintained prominent patency with the mono-layer ciliated epithelium and demonstrated minimal OAD manifestations.While in the groups with normal saline and culture solutions for comparison,obvious OAD manifestations were present.(There were obvious OAD manifestations in the groups with normal saline and culture solutions.) The CD4+/CD8+ ratio was 1.5±0.3 in the allografts with epithelial implantation;1.6±0.3 in grafts with normal saline inoculation.Conclusions:Both antigen expression and integrity maintenance of epithelial cells are indispensably important in OAD development.An integrated epithelium from ex-vivo culture was able to suppress submucous fibroproliferation and maintain luminal patency.Moreover,epithelial implantation was a possible method for fulfilling the process of re-epithelialization.Chapter Four The integrity and barrier function of the new epithelial layer of exo-genic origin were examined.Objective:To study the integrity and barrier function of the new epithelial layer of exo-genic cellular origin.Materials and Methods:There were 60 SD rats and Wistar rats included in this experiment.After ex-vivo culture,SD rat-origin epithelial cells were inoculated into the tracheal segments of nude Wistar rat,which were then heterotopically transplanted into the recipient SD rats.Four weeks after transplantation,the allografts underwent histopathological and ultrascopic examinations,as well as histochemical stain of epithelial CK14,CK18,CFTR and ZO-1 expressions.Results:The new epithelium was composed of prominent ciliated epithelial cells which had well preserved the organelles and abundant mitochondria.There were plenty of intercellular tight junctions. CK14,ZO-1 and CFTR expressions were positive in this new epithelium.Conclusions: The barrier function was restored in the new epithelial layer of exo-genic epithelial origin. Epithelial implantation is a possible method for tissue-engineering donor trachea modification with the recipient of epithelial phenotype.Summarization①Rodent orthotopic and heterotopic tracheal transplantations can induce distinctive OAD manifestations,and are,therefore,suitable for OAD study.②Epithelium plays a key role in OAD development,which can be reasoned from the different pathologic outcomes of the above two tracheal transplantation models.③Epithelial phenotype re-modulation is the key point in maintaining the donor tracheal luminal patency.④Epithelial implantation with the seed cells from ex-vivo culture can have protections against luminal obliterations.⑤Epithelial implantation can lead to formation of an integrated epithelium,which also has a better preserved barrier function.更多还原