【摘要】：背景和目的：先天或后天等因素可導(dǎo)致尿道缺損,臨床上尤其以尿道下裂最為常見,對于這些患者,常常需要進(jìn)行尿道重建,傳統(tǒng)外科重建的方法常常導(dǎo)致許多并發(fā)癥,包括：尿道皮膚瘺,尿道狹窄等。復(fù)雜的尿道狹窄仍是泌尿外科最難解決的問題之一。尿道狹窄發(fā)生的重要原因是術(shù)后尿道缺少粘膜的保護(hù),導(dǎo)致疤痕形成,進(jìn)而形成狹窄。尿道手術(shù)的成功關(guān)鍵之一是尿道粘膜快速生長。以往許多材料被研究者用來修復(fù)尿道缺損,如膀胱粘膜、包皮、舌粘膜、頰粘膜以及結(jié)腸粘膜等。但是,這些方法并發(fā)癥多,且都是犧牲一個(gè)器官來重建另一個(gè)器官。組織工程技術(shù)可能為尿道重建提供一種新的方法。本研究的目的是利用骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)、平滑肌細(xì)胞(smooth muscle cells, SMCs)和膀胱脫細(xì)胞基質(zhì)(bladder acellular matrix, BAM)構(gòu)建組織工程片狀移植物(tissue-engineered sheet graft, TESG),在兔模型上驗(yàn)證其修補(bǔ)尿道缺損的可行性。 方法：體外培養(yǎng)擴(kuò)增BMSCs和SMCs,制作兔BAM,先將1×106BMSCs種植于BAM上孵化3天,再將1×106SMCs種植于BAM的另一面上孵化3天,構(gòu)建TESG;將構(gòu)建好的TESG移植到網(wǎng)膜包裹2周,另設(shè)立沒有種植細(xì)胞的BAM移植宿主到網(wǎng)膜內(nèi)包裹2周為對照組。構(gòu)建尿道缺損動(dòng)物模型,尿道缺損長約3cm,寬約1.5cm,缺損部位距離尿道外口約2cm。而后利用TESG修復(fù)尿道缺損。取實(shí)驗(yàn)組24只雄兔共分四組,每組6只,術(shù)前禁食8小時(shí)；另取6只作為對照組,采用未種植細(xì)胞的BAM修復(fù)尿道缺損。手術(shù)前均留置導(dǎo)尿,于術(shù)后兩周左右拔除。標(biāo)本分別于手術(shù)后2、4、8、16周采集,行HE染色,采用免疫組化檢測AE1/AE3、uroplakinⅢa、ZO-1,以及平滑肌的生長情況。 結(jié)果：BMSCs三天后能貼壁,7天后細(xì)胞快速增殖,呈線性融合,做流式細(xì)胞儀檢測顯示BMSCs高表達(dá)CD29(98.5%)、CD44(99.6%)和CD90(99.7%),而不表達(dá)CD34(4.0%)。對BAM作HE染色和掃描電鏡顯示未見明顯的細(xì)胞殘片。將TESG種植到網(wǎng)膜包裹兩周后作HE檢測顯示：TEST表面有薄層上皮生長。動(dòng)物實(shí)驗(yàn)中,直到取材時(shí)所有的動(dòng)物均存活,術(shù)后2周切口都已痊愈,拔除尿管后未見明顯尿道皮膚瘺發(fā)生。術(shù)后8周移植物和尿道之間沒有明顯的界限。術(shù)后2周,HE染色顯示TESG完全被薄層上皮覆蓋,TESG和正常組織的界限仍很清楚；到術(shù)后4周,TESG被覆上皮變厚,可見平滑肌生長。到第8周及16周,TESG有多層上皮覆蓋。免疫組化顯示抗AE1/AE3、抗uroplakinllla、和抗ZO-1均陽性,也進(jìn)一步證實(shí)了上皮的再生情況。對照組中3只兔子在術(shù)后4周內(nèi)均死亡,尸檢顯示尿道完全狹窄；對照組中另三只兔子排尿明顯變細(xì),顯示有不同程度的尿道狹窄。 結(jié)論：利用BMSCs、SMCs和BAM構(gòu)建TESG修復(fù)尿道缺損是可行的, BMSCs有可能促進(jìn)尿路上皮的再生。
[Abstract]:Background and objective: urethral defects can be caused by congenital or acquired factors, especially hypospadias. Urethral reconstruction is often needed in these patients. Include: urethral skin fistula, urethral stricture and so on. Complex urethral stricture is still one of the most difficult problems in urology. The main cause of urethral stricture is the lack of mucosal protection, resulting in scar formation. One of the keys to the success of urethral surgery is the rapid growth of urethral mucosa. Many materials have been used to repair urethral defects, such as bladder mucosa, prepuce, tongue mucosa, buccal mucosa and colon mucosa. However, these methods have many complications and all sacrifice one organ to rebuild another. Tissue engineering may provide a new method for urethral reconstruction. The aim of this study was to construct tissue engineered flake grafts (tissue-engineered sheet graft, TESG),) from bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs),) smooth muscle cells (smooth muscle cells, SMCs) and bladder acellular matrix (bladder acellular matrix, BAM). The feasibility of repairing urethral defect was verified on rabbit model. Methods: rabbit BAM, was incubated with 1 脳 106BMSCs on BAM for 3 days, then 1 脳 106SMCs was incubated on the other side of BAM for 3 days. TESG; was constructed by using amplified BMSCs and SMCs, in vitro. The constructed TESG was transplanted into the omentum for 2 weeks, and the BAM graft host without implanted cells was set up as the control group for 2 weeks. An animal model of urethral defect was established. The urethral defect was about 3 cm in length and 1.5 cm in width. The defect was located about 2 cm from the external urethral orifice. TESG was then used to repair urethral defects. 24 male rabbits in the experimental group were divided into four groups, 6 rabbits in each group, fasting for 8 hours before operation, and 6 rabbits as control group. The urethral defects were repaired by BAM without implantation cells. Catheterization was performed before operation and was removed about two weeks after operation. The specimens were collected at 816 weeks after operation and were stained with HE. The growth of AE1/AE3,uroplakin 鈪，

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