【作者】 吴明明；

【导师】 崔鹏程；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2008， 硕士

【摘要】 临床上由于各种喉气管疾病如外伤、肿瘤等因素导致的喉气管狭窄,需切除狭窄段并修复之。通常,气管缺损如达成人气管1/2及儿童气管1/3长度,可行端端吻合术来修复;当超过这个界限时,需植入气管假体才能重建气管的连续性。自体组织移植如肋软骨、鼻中隔软骨、胸锁乳突肌等,因增加创伤、供体来源有限等原因使用受限。同种异体材料及人工材料也存在着感染、远期排斥等问题而难以实用。因此有效的气管替代材料的应用是十分重要的。随着组织工程技术的迅猛进展,气管组织工程的研究也逐渐受到重视。气管重建成功的关键环节有三:1、软骨支架重建;2、创面在瘢痕形成之前再上皮化;3、移植物的再血管化。经过多年努力,软骨支架的构建已在裸鼠体内获得成功。然而,理想的气管替代物必需考虑气管管腔内衬以有功能的气管上皮细胞,且良好的血管化也是保持结构成分的可活性的重要因素。但是,目前国内在气管上皮细胞的培养技术方面尚未成熟,有待于进一步研究。细胞支架的选择也是非常重要的。胶原不仅支持组织重建,而且有利于多种细胞在体外培养,同样也适合于上皮细胞的形成和分化,有报道使用胶原凝胶培养大鼠、豚鼠、兔、狗和人的气管上皮细胞?[1-3]?。SIS是天然细胞外基质类生物材料,一般取自猪的小肠粘膜下层。近年来,SIS作为一种生物支架材料在组织工程研究中日益受到重视,并广泛应用于骨骼肌、腹壁、硬脑膜、膀胱、血管等组织器官缺损的修复?[4]?。有报道将SIS复合上皮细胞及成肌细胞用来构建组织工程食管?[5]?。当前,用于软骨组织工程支架的合成材料主要是聚酯类的PGA和PLA,以及PGA和PLA的共聚物。既往我科使用鼻中隔软骨细胞种植到PGA无纺网支架,在裸鼠体内形成了管状软骨组织。本研究以胶原复合SIS及PGA无纺网支架材料分别复合气管粘膜上皮细胞和软骨细胞进行体外培养,再将双层复合物相叠加形成管状结构,埋植入裸鼠体内培养,通过观察动物存活时间及移植物体内演变情况,为复合气管支架的构建提供理论和实验依据。研究目的1.探讨建立兔气管粘膜上皮细胞的原代培养方案,自制兔小肠粘膜下层(SIS),通过胶原、SIS与气管粘膜上皮细胞共培养,观察气管粘膜上皮细胞与SIS的组织相容性,为使SIS作为上皮组织工程化载体打下基础。2.评估软骨细胞与PGA载体的组织相容性及PGA及SIS两种支架材料体内降解的情况。3.观察裸鼠存活时间及移植物体内演变过程,明确构件的生长及血管结构的形成情况。材料与方法1、兔气管粘膜上皮细胞培养及其与自制小肠粘膜下层复合培养的实验研究应用组织块法培养兔气管粘膜上皮细胞,无血清培养基体外培养,倒置显微镜观察细胞生长分化情况,抗角蛋白单克隆抗体免疫组织化学染色及扫描电镜验证纤毛上皮细胞特性。用顺序化学脱细胞处理法机械剥离获取兔小肠粘膜下层,进行组织学检测。将气管上皮细胞与胶原、SIS体外复合培养,行组织学观察。2、原代软骨细胞-PGA共培养及体外构建管状气管支架用胰酶消化法获得原代法兔关节软骨细胞,行倒置显微镜及组织学观察。再将软骨细胞种植在PGA无纺网支架上体外培养1w,将上述气管粘膜上皮复合物叠加在软骨细胞-PGA层上,包绕在直径4mm实心玻璃棒上,可吸收缝合线捆绑固定。3、复合气管支架体内培养的实验研究将复合管状气管支架分别埋植于9只裸鼠前肢腋下,观察裸鼠存活时间及构件在动物体内演变过程;于4、8、12w后分批处死动物,每批3只,取出构件进行组织学鉴定,观察其粘膜上皮、软骨以及血管结构的形成情况。结果1、兔气管粘膜上皮细胞培养及其与自制小肠粘膜下层复合培养的实验研究原代气管上皮细胞培养1w后增殖旺盛,10d时上皮细胞分布范围更广,并汇合呈铺路石样生长,部分细胞可见纤毛活动,抗角蛋白单克隆抗体免疫组织化学染色及扫面电镜验证纤毛上皮细胞特性。将气管上皮细胞-SIS复合物体外培养1w后,组织学检测示:可见气管粘膜上皮和SIS双层结构形成,气管粘膜上皮较薄,其下的SIS层较厚,呈较均匀的结缔组织基质。胶原层不明显。2、原代软骨细胞-PGA共培养及体外构建管状气管支架原代软骨细胞活性较强,增殖旺盛,约1w细胞基本长满瓶壁,组织学检测显示软骨细胞特性。将其种植于PGA支架形成软骨细胞-PGA复合物,再将气管粘膜上皮复合物叠加在其上,将叠加物包绕在直径4mm、长7mm的实心玻璃棒上,可吸收缝合线捆绑固定,可以在体外构建出管状气管结构。3、复合气管支架体内培养的实验研究将气管支架种植到裸鼠体内后,观察整个实验过程未见构件的感染和排出。分批处死动物后,取出标本,移除玻璃棒,大体观察可见:4w时移植物大小与植入前基本一致;8w时外形较前略有缩小,可见瓷白色软骨样结构,触之有韧性,管腔基本完整;12w时形态与8w时基本相同。HE染色石蜡切片,光镜观察示:4w时气管上皮及软骨细胞结构尚不典型,仍有较多的PGA纤维和SIS残留。8w时,开始出现椭圆形软骨陷窝,软骨细胞间距加大,近管腔面可见纤毛样上皮结构形成,但尚不完整;P GA纤维基本降解吸收,但SIS支架仍有残留。12w时整体组织结构较8w时成熟;软骨层表面覆盖一薄层粘膜上皮结构,未观察到粘膜下血管结构形成;SIS基本完全降解吸收。结论1.组织块法培养的原代气管粘膜上皮细胞具有一定的增殖能力,此方法对获取组织工程种子细胞具有一定的应用价值,但由于取材量少,一次不能得到大量的气管上皮细胞,故尚不能满足气管修复实验的要求。顺序化学脱细胞法制备的SIS安全性好,易保存,操作性强,可重复制备,且细胞相容性好,故可以作为气管上皮细胞理想的支架材料。2. PGA有良好的细胞相容性,软骨细胞在其表面生长,不影响细胞的形态,因此是较好的软骨细胞载体。PGA和SIS材料在体内约8～12w可降解吸收。3.复合气管支架植入裸鼠体内8w及12w时,组织学检测显示粘膜上皮和软骨结构的形成,但粘膜下未见明显的血管结构,故移植物的血管化尚有待于进一步研究。更多还原

【Abstract】 It is often necessary to resect laryngotracheal stenosis and to reconstruct theresulting defects for patients who have various types of tracheal disease liketrauma or tumor that cause stenosis.Circumferentially resected tracheas are conventionallyreconstructed by endtoendanastomosis,but the general limits of saferesection are about half of the tracheal length in adults and probably one third insmall childen.Tracheal resection longer than these limits requires replacement.Using autogenous tissue such as costal cartilage,nasal septal cartilage to patch thedefects takes risk of adding wounds and the source of grafts is lmited.Allograftsand artificial materials are also limited because of infection and longtermrejection.Therefore,effective production of grafts for tracheal reconstruction is very important.As tissue enginnering technique develops quickly,tissueengineeredtrachea have been thought highy of increasingly.The key to success fortracheal reconstruction is to reconstruct cartilage stent,reepithelization andrevascularization.Now,the cartilage stent has been made in nude mousesuccessfully.But functional tracheal epithelium and good vascularization are stillbig problems.Choosing cell stent materials is also very substaintial.Collagen is importantnot only for supporting tissue construction,but also for many kinds of cells beingcultured in vitro. Collagenous gel is also suitable for differentiation and formationof epithelial cells.With this method,culture of tracheal epithelial cells of severalspecies,such as rats,guinea pigs,rabbits,dogs,and humans has been reported [13].SIS is a nature,biocompatible,acellular,collagenbasedmatrix derived fromthe porcine small intestine submocosa. For the past few years,SIS has beenwidely used for the repair of skeletal muscle, abdominal wall, dura matermembrane,bladder,and vessels [4] .Also,some researcher fabricated the frameworkof a biodegradable artificial esophagus with the epithelial cells and the myoblastcells seeded on the small intestinal submucosa [5] .PGA,PLA and the copolymer ofthe both are main synthetic materials of cartilage tissueengineeringstents. In ourprevious studies,we demonstrated that human nasoseptal chondrocytesPGAconstruct could develop into a new cartilage in predetermined shapes in athymicmice.In this study,we would seeded primary rabbit tracheal epithelial cells andcartilage cells onto the surface of SIS covered by collagen and a nonwoven meshof polyglycolic acid(PGA) respectively to form epitheliumSISand chondrocytesPGAconstructs.Then the two layers were stratified to make tubershaped structure,which was implanted into the donor of athymic mice to grow. By investigatingthe compound grafts’growth in vivo and animals’survival time,we cansupply theoretical and experimental evidence for the construction of compoundtracheal framework.Objective1. To investigate an appropriate model of rabbit tracheal primary epithelial cellculture.To find suitable way to make SIS as scaffold for tissue engineering.Toinvestigate the biocompatibility of SIS with epithelial cells and to explore thepossibility to construct tissue engineering epithelium with SIS as the scaffold andepithelial cells as the seed cells.2. To evaluate the biocompatibility of PGA with cartilage cells and thedegradation of the two stents of PGA and SIS in vivo.3. To investigate compound grafts’variation in athymic mice and animals’survival time,and identify the grafts’growth and formation of blood vessels.Materials and methods1. Model of tracheal epithelial cells primary culture and selfmadeSISpreparation and coculturewith epithelial cellsRabbit tracheal ciliated epithelial cells were obtained by an explantoutgrowth culture system,cultured with serumfreemedium supplementedhormones and growth factors in vitro,observed by photocontrast microscopy.Antikaratin monoclonal antibody immunohistochemical stain and scanningelectron microscope(SEM) were employed to verify the characteristics ofepithelial cells.Derived from rabbit jejunum,SIS was dealt with steps chemicaldecellulaand examined by histological stain.Then the SIS taken from rabbits was mixed with epithelial cells taken from homogeneous rabbits for culture invitro to form epitheliumSISconstruct.Histological observation was done underphase contrast microscope.2. Coculture of primary chondrocytes with PGA and construction of tubershapedof tracheaCartilage cells obtained from rabbit joint cartilage by enzyme digestion werecultured in vitro.After 1 week,the cells were characterized by photocontrastmicroscopy and also examined with hematoxylineosinstaining and AB/PASstaining.Then the cells were seeded onto a nonwoven mesh of PGA to form acellPGAconstruct. Then epitheliumSISlayer was stratified onto chondrocytesPGAlayer to form a compound, and the compoumd was wrapped around adiameter of 4 millimeter glass rod, and bound by absorbable suture.3. Compound tracheal construction cultured in vivoThe compound grafts were implanted into forelimb oxter of 9 athymicmice separately.The grafts’growth in vivo and animals’survival time wereobserved. The specimens were harvested 4 ,8,12 weeks after implantation andsubjected to gross morphologic and histologic analysis.Results1. Model of tracheal epithelial cells primary culture and selfmadeSISpreparation and coculturewith epithelial cellsMany tracheal epithelium cells with good proliferation property were foundin the culture system after 1 week. Epithelial cells distributed widely andconfluented at 10th day.During that time ciliary beating was active,and theverification tests presented positive. HE staining showed the two layers of theepithelium and SIS.Collagen was not evident. 2. Coculture of primary chondrocytes with PGA and construction of tubershapeof tracheaPrimary cartilage cells was very active and with good proliferationproperty.The cells confluenced in about 1 week,and the histological stainingverified cartilage cell characteristics.Then the cells were seeded onto a nonwovenmesh of PGA to form a cellPGAconstruct. The construct with epitheliumSISlayer above could make tubershapein vitro by wrapping around a diameter of4 millimeter and lenghth of 7 millimeter glass rod.3. Compound tracheal construction cultured in vivoIn vivo study,no evident inflammatory infection and rejection of the constructsafter implantation were found.After animals were killed in batch, the glassrods were taken out of the specimens. The gross specimens of 4 weeks revealedapproximately the same shapes as original predetermined shapes.At 8 and 12weeks newlyformedcartilage had fair elasticity and support ability,and luminaswere almost integrated. Histological observation demonstrated that new cartilagecells and tracheal epithelial layer were not typical yet in 4 weeks. In 8 weeks,thecolumnar ciliated epithelium was observed towards the face of the lumina but notintegrated,and the PGA were almost degradated.Until in 12 weeks,the SIS of thespecimens were nearly absorbed.At the time,there was an epithelium mucosaeabove the cartilage layer,but the structure of blood vessels could not be foundunder the tracheal mucosa in the whole study.Conclusion1. The explant outgrowth culture system may be useful in establishing a culturesystem for ciliated cells. In this way, using cultured ciliated epithelial cells asseeds for tracheal tissue engineering was potentially.Steps chemical decellular method was a good way made SIS as scaffold for tissue engineering because ofits security,easy to conservation and remake,and operability.SelfmadeSIS can beused as the scaffold to construct tissue engineering epithelium as it has goodbiocompatibility with tracheal epithelial cells.2. PGA also has good biocompatibility with cartilage cells without disturbing thecells form or inhibiting the growth and function of cartilage cells,so it can be agood kind of tissueengineeringcartilage scaffold.PGA and SIS can be degradatedin 8 and 12 weeks in vivo.3. After the grafts were embedded into athymic mice in 8 and 12 weeks,trachealepithelium and cartilage could be observed in histological staining but nostructure of blood vessels could be found under the tracheal mucosa.Therevascularization of the constructs should be further studied for longtermapplication.更多还原