【作者】 陈伟豪；

【导师】 刘春晓；

【作者基本信息】 南方医科大学 ， 泌尿外科学， 2008， 硕士

【摘要】 【研究背景】尿道重建术中,因先天因素、损伤、炎症或肿瘤等原因造成尿道缺损的治疗一直是泌尿外科的难题,目前文献报道临床治疗方法多种多样,但其修复多用自身其他组织替代,如用自体皮肤粘膜、膀胱粘膜、口腔粘膜或阑尾等,或者游离松解尿道断端后行端端缝合;但这些方法以牺牲正常组织为代价,手术创伤大,应用自身组织更加重患者的创伤,导致术后并发症居高不下,效果不甚理想,所以寻找一种全新的方法成为当前的热点。新兴的组织工程方法是尿道缺损修复的一个方向,并取得了突破性进展。从最初1971年Court等用硅胶管进行尿道替代的动物试验,结果不令人满意,但为尿道替代材料的研究开创了新局面。Italiano等用一种由透明质酸合成的可降解的支架材料来修复兔尿道,7个月后尿道放射学检查见移植物消失,尿道重建,再生组织在一定压力下具有良好的伸展性,组织学观察见再生尿道组织中有过度的上皮细胞形成,并有丰富的弹性纤维。最初的研究采用在人工合成细胞支架上种植尿道粘膜及平滑肌细胞修复尿道缺损,近年来多用小肠粘膜下层(SIS)、膀胱粘膜下层(BAMG)或脱细胞基质支架(ECM),Sievert等用同种异体脱细胞尿道基质修复兔长达1 cm左右的缺损,术后10天,上皮细胞从宿主迁移至基质支架,术后3周基质支架的尿道管腔完全由上皮细胞覆盖,3个月时可见平滑肌细胞束长入到基质,6个月时平滑肌细胞束覆盖了三分之一的基质支架,尿道动力学示实验组及对照组无差别,8个月时行尿道造影未能辨别宿主及移植物,他们认为该材料可用于一期尿道重建。傅强等利用脱细胞膀胱粘膜下筋膜修复分别修复兔的尿道1cm左右及2-3cm缺损,其中1cm缺损修复效果良好,他们也认为单纯用细胞外膀胱粘膜下筋膜无法修复2-3cm以上缺损。目前尿道组织工程研究常用的细胞支架有人工合成细胞支架、天然材料细胞支架及脱细胞基质支架。尿道组织工程虽然取得了很大进步,但尚未找到理想的细胞支架,仍有大量问题需要解决,存在问题:①人工合成细胞支架的组织相容性较差,引起不同程度的炎症反应;②天然材料细胞支架及脱细胞基质支架来源缺乏,加工能力有限,耗资高,难以形成产业化,导致价格昂贵;③延展性、伸缩性等机械性能方面尚无法满足临床应用。因此研究生物性能好、价格低廉、原料来源丰富的细胞支架是组织工程学的一个重要课题。科研人员一直致力于研究、探索开发新型的细胞支架,主要在原材料、制作工艺、支架材料改性及表面修饰等方面寻找突破点,越来越多的材料进入研究人员的筛选范围。近年来,蚕丝作为一种天然的高分子材料受到人们的广泛重视,其提取物丝素蛋白是蚕丝的重要组成成分,具有无毒性、无刺激作用,对机体细胞粘附能力强,具有良好的物理化学性能和生物相容性,以丝素蛋白为原料制成的组织工程细胞支架、手术缝线、隐形眼镜、人工血管、皮肤创面保护膜、硬脑膜修补材料效果良好,在生物医学领域得到广泛的应用。刘春晓及林阳彦等将丝素蛋白膜作为细胞支架用于修复兔子尿道1.5cm长度缺损取得良好的效果,但不同长度缺损修复效果尚不明确。【目的】本研究旨在探索丝素蛋白膜修复不同长度尿道缺损的效果【材料与方法】1.动物和材料:成年雄性犬(10.0—12.9Kg)14只(广东肇庆市实验动物科技研究中心提供);丝素蛋白膜:孔径150～250μm,孔隙率65%～95%(本材料由浙江大学闵思佳教授惠赠);广谱细胞角蛋白单克隆抗体(PCK)(博士德公司)。2.动物分组:14只犬随机分成3组:实验组Ⅰ(n=6),实验组Ⅱ(n=6),对照组(n=2)。3.实验方法:3.1实验组Ⅰ:游离出尿道海绵体,将尿道海绵体段分别切除1.5cm,再用1.5cm长度丝素蛋白支架修复尿道缺损;实验组Ⅱ:游离出尿道海绵体,将尿道海绵体段分别切除3.0cm,再用3.0cm长度丝素蛋白支架修复尿道缺损。3.2对照组:行尿道海绵体游离后即逐层缝合海绵体包膜、皮下组织及皮肤;4.检测方法:组织再生情况病理切片观察及逆行尿道造影4.1实验组Ⅰ及实验组Ⅱ分别在术后6周、12周每次各取1只行逆行尿道造影,然后处死取材行苏木精—伊红(HE)染色、Van Gieson(VG)染色及免疫组织化学染色观察组织再生情况;4.2对照组分别于6周及12周每次各取3只行逆行尿道造影,然后处死取材行苏木精—伊红(HE)染色、Van Gieson(VG)染色及免疫组织化学染色观察组织再生情况;【结果】1.实验中犬存活及手术切口愈合情况:14只犬实验过程中无一例死亡。2.逆行尿道造影示:实验组Ⅰ6只动物术后尿路造影未见尿瘘及明显狭窄,修复区6周时修复区稍粗糙、不光滑,术后12周后与对照组无明显差异;实验组Ⅱ6只动物均出现尿瘘或管腔狭小、部分充盈缺损,术后6周和12周无明显差异。3.组织标本肉眼观察:对照组尿道无缺损及瘢痕形成,无尿道狭窄;实验组Ⅰ1.5cm修复区术后6周可见少许瘢痕增城,尿道稍狭窄,12周较6周改善;实验组Ⅱ3.0cm修复术6周瘢痕形成,可见缺损,组织质地坚韧,12周与6周无明显差别。实验组所有动物修复区丝素蛋白膜均固定于原位,术后6周丝素蛋白膜已部分降解,组织均匀覆盖,12周修复区丝素蛋白膜已难于分辨。4.组织学检查:苏木精—伊红(HE)及Van Gieson(VG)染色4.1对照组术后6周及12周尿道组织结构无明显区别,尿道上皮细胞均匀覆盖,层数约4—5层,排列规则,未见炎症细胞,粘膜下可见较多的平滑肌细胞,无纤维化形成。4.2实验组Ⅰ1.5cm修复区术后6周可见少量炎症细胞,丝素蛋白支架表面粘附细胞排列不均匀,层数约6—9层,同时可见较多的成纤维细胞及胶原纤维;12周尿道上皮细胞排列均匀,完全覆盖,层数约4—5层,平滑肌细胞增多,成纤维细胞及胶原纤维较6周时减少,排列规则,无明显炎症细胞浸润,可见血管口径增大,与对照组相似。4.3实验组Ⅱ3.0cm修复区6周时腔壁纤维化形成,可见大量的纤维组织及成纤维细胞,大部分区域上皮细胞缺失;12周仍以纤维组织为主,瘢痕化形成,可见陈旧性纤维组织,上皮细胞增多,排列混乱,偶可见少量平滑肌细胞,与6周时无明显差别。5.免疫组织化学检查:应用广谱细胞角蛋白单克隆抗体染色,对照组尿道上皮细胞呈阳性棕黄色,高倍镜观察可见胞浆内棕黄色网状角蛋白颗粒,排列规则,术后6周和12周没有明显区别;实验组Ⅰ1.5cm修复区术后6周可见上皮细胞生长,术后12周后趋于规整,与对照组无明显差别;实验组Ⅱ3.0cm修复区术后6周大部分区域上皮细胞缺失,少数区域可见少量上皮细胞,术后12周时上皮细胞未见明显增加,与术后6周无明显区别。【结论】丝素蛋白膜能够诱导尿道粘膜上皮细胞及尿道平滑肌的生长,具有促进尿道缺损修复的能力。但单纯丝素蛋白膜只能修复1.5cm短距离尿道缺损,对3.0cm以上长距离效果不理想。更多还原

【Abstract】 BackgroundReconstruction of urethral defect has always been a difficult problem for urology owing to congenital factor,trauma,inflammation or tumor.So far,as having been reported,the methods of clinical treatment are various.Most of treatments for repairing urethral defect are using other tissues,such as membrane of skin,appendix, mucous of bladder and mouth and so on.However,these treatments would cause a damage to normal tissues,bring an extra physical injury and have more complications. From the above mentioned,we can see that the effects of these treatments are far from perfect.It’s the most important thing to seek a new way to solve the problem.Tissue engineering shows a bright future for repairing urethral defect,and has gained a breakthrough.Court Replaced a urethral segment with a silicone elastomer tube in the maledog in 1971,it was a new beginning thought the experiment failed.Italiano observed that using hyaluronic acid to replace the defective urethra section in the rabbits would help the defetive urethra to regenerate the construction well.In the initial researches,urethral defect were repaired with synthesized cells scaffold in which urethral epitheliums and smooth muscle cells were seeded,but recently usually repaired with SIS(Small intestinal submucosa),BAMG(Bladder acellular matrix graft) or ECM(Extracellular matrix).Sievert indicated homologous acellular matrix graft will help for urethral reconstruction in the rabbit.FuQiang used acellular matrix succeed in repairing the lcm defective section,but failed in repairing the 2-3cm defective section.The cells scaffolds which are mainly used in tissue engineering at present include synthesized cells scaffold,scaffold made with natural material and ECM.Though a great progress has been made in tissue engineering, there are still many problems,such as the shortage of perfect scaffold.The reasons are as follows:①synthesized cells scaffold brings inflammation for its poor biocompatibility;②as to the natural cells scaffold,for its rare material,limited processing ability,it also hard to get into industrialization,all the above lead to a expensive price;③the mechanical properties,such as ductibility and retractility can’t suit to the clinical treatment.Therefore,how to get perfect scaffold with good biocompatibility,lower cost and abundant source is an important task on relevant researches.The researchers have been studying and exploring new cells scaffold, mainly in terms of raw materials,processing technique,surface modification,and so on.As a kind of macromolecule material,silk has been taken more and more attention in recent years.It has been confirmed that silk fibroin,a key component of silk,has no stimulation,but avirulence,strong adherence,good physical and chemical character and biocompatibility.Silk fibroin has been used in biomedical field widely, such as making cells scaffold,artificial vessel,protective membrane for wounded skin and endocranium repairing material.Liu Chunxiao and Lin Yangyang reported that using silk fibroin can repair the 1.5cm defect in rabbit urethra,it’s unclear that the length of urethral defect can be repair only by silk fibroin.ObjectivesTo estimate the effect of different length of urethral defect repaired with Silk fibroin filmMethods1.0)Animal and material:14 maledogs(10.0—12.9Kg)(GUANGDONG ZHAOQING LABORATORY ANIMALS RESEARCH CENTER);Silk fibroin film:aperture 150～250μm,interval porosity 65%～95%(presented by professor Min Si-Jia of Zhejiang University);Mouse Anti—Pan Cytokeratin(PCK) antibody (BOSTER BIOTECHNOLOGY CO.LTD.).2.0) 14 Male maledogs were divided into three groups randomly:experimental groupⅠ(n=6),experimental groupⅡ(n=6) and control group(n=2).3.0) Experimental method:3.1) In the experimental groupⅠwith a defect of 1.5cm while groupⅡwith a defect of 3.0cm in the urethra in each maledog,all of them were repaired with Silk fibroin film;3.2) Control group of 2 maledogs underwent a sham operation;4.0) Detecting method:Urethra reorganization and retrograde urethrography4.1) The maledogs of the control group underwent retrograde urethrography and then the specimens of regenerative urethral obtained at 6 and 12 weeks postoperatively were observed by histological technique(HE and VG staining) and immunohistochemistry respectively.4.2) The maledogs of the experimental group underwent retrograde urethrography and then the specimens of regenerative urethral obtained at 6 and 12 weeks postoperatively were observed by histological technique(HE and VG staining) and immunohistochemistry.Results1.The survival of the maledogs and the heal condition of operative incision during experiment:None of the 14 maledogs died during experiment.2.The observation of the specimens of regenerative urethral with naked eye: The control group had no cicatrices formation and stricture and there were no obvious difference between 6 weeks and 12 weeks after the operation.The experimental groupⅠwith 1.5cm defective section had a little cicatrices formation or stricture 6 weeks post operation,12 weeks after operation tend to be better than 6 weeks;fistula or strictures were observed in all of the animals in experimental groupⅡwith 3.0cm defective section,and there were no obvious difference between 6 weeks and 12 weeks after operation.The Silk fibroin film of the experimental group fixed in the original place and 6 weeks later,the Silk fibroin film partly degradated and the surface of the Silk fibroin film was covered by a little organization uniformly;12 weeks,Silk fibroin film was difficult to identify and there was no obvious difference with control group.3.Retrograde urethrography:All the experimental groupsⅡhad stricture or urinary fistula while the all control groups and the experimental groupⅠdidn’t have.4.Histological examination:Hematoxylin-eosin(HE) and Van Gieson(VG) staining.4.1 The urethral organization of control group had no obvious difference between 6 weeks and 12 weeks after operation.About 4-5 layers urethral epitheliums covered uniformly and regularly,There was no inflammatory cell or fibrosis under the membrane and many smooth muscle cells were observed..4.2 In the experimental groupⅠof the 1.5cm defective section there were large amount of inflammatory cells 6 weeks after operation,containing neutrophil, lymphocyte,lymphomonocyte and eosinophile,and besides there were 6-8 layers of urethral epitheliums covered unregularly,some smooth muscle cells and small vessels were visible occasionally,many collagenoblasts and collagen fibers were observed;12 weeks,the urethral epitheliums arranged uniformly and gradually,about 4-5 layers,smooth muscle cells increased,collagenoblasts and collagen fibers decreased and arranged gradually,caliber of the blood vessels enhanced,there was no obvious difference from control group.4.3 In experimental groupⅡof the 3.0cm defective section,6 weeks after operation there were large amount of inflammatory cells,containing neutrophil, lymphocyte,lymphomonocyte and eosinophile,and besides little urethral epitheliums,smooth muscle cells and small vessels were visible occasionally,many collagenoblasts and collagen fibers were visible;12 weeks latter,the urethral epitheliums,smooth muscle and small vessels didn’t increased,collagenoblasts and collagen fibers remaind the same as 6 weeks ago.There were no obvious difference between 6 and 12 weeks after operation.5 Immunohistochemistry examination:Dyeing with Mouse Anti—Pan Cytokeratin(PCK) antibody,the urethral epitheliums of control group indicated positive,arranging uniformly,no obvious difference between 6 and 12 weeks;In the experimental groupⅠof the 1.5cm defective section indicated positive and the arrangement of the epitheliums tend to be more and more regularly from 6 weeks to 12 weeks.In experimental groupⅡof the 3.0cm defective section indicated seldom areas were covered by epithelium after 6 weeks.12 weeks after the operation,the epitheliums didn’t increased obviously.Conclusion:Silk fibroin film appears to promote the urethral defect repair,which induces the growth of urethra epitheliums and smooth muscle cells.It can repair short length defect within 1.5cm,while it is unpredictable to repair defect longer than 3.0cm only by the materials.更多还原