利用骨髓间充质干细胞、平滑肌细胞和膀胱脱细胞基质构建组织工程尿道的研究

发布时间：2018-12-20 07:24

【摘要】：背景和目的：先天或后天等因素可导致尿道缺损,临床上尤其以尿道下裂最为常见,对于这些患者,常常需要进行尿道重建,传统外科重建的方法常常导致许多并发症,包括：尿道皮肤瘘,尿道狭窄等。复杂的尿道狭窄仍是泌尿外科最难解决的问题之一。尿道狭窄发生的重要原因是术后尿道缺少粘膜的保护,导致疤痕形成,进而形成狭窄。尿道手术的成功关键之一是尿道粘膜快速生长。以往许多材料被研究者用来修复尿道缺损,如膀胱粘膜、包皮、舌粘膜、颊粘膜以及结肠粘膜等。但是,这些方法并发症多,且都是牺牲一个器官来重建另一个器官。组织工程技术可能为尿道重建提供一种新的方法。本研究的目的是利用骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)、平滑肌细胞(smooth muscle cells, SMCs)和膀胱脱细胞基质(bladder acellular matrix, BAM)构建组织工程片状移植物(tissue-engineered sheet graft, TESG),在兔模型上验证其修补尿道缺损的可行性。 方法：体外培养扩增BMSCs和SMCs,制作兔BAM,先将1×106BMSCs种植于BAM上孵化3天,再将1×106SMCs种植于BAM的另一面上孵化3天,构建TESG;将构建好的TESG移植到网膜包裹2周,另设立没有种植细胞的BAM移植宿主到网膜内包裹2周为对照组。构建尿道缺损动物模型,尿道缺损长约3cm,宽约1.5cm,缺损部位距离尿道外口约2cm。而后利用TESG修复尿道缺损。取实验组24只雄兔共分四组,每组6只,术前禁食8小时；另取6只作为对照组,采用未种植细胞的BAM修复尿道缺损。手术前均留置导尿,于术后两周左右拔除。标本分别于手术后2、4、8、16周采集,行HE染色,采用免疫组化检测AE1/AE3、uroplakinⅢa、ZO-1,以及平滑肌的生长情况。 结果：BMSCs三天后能贴壁,7天后细胞快速增殖,呈线性融合,做流式细胞仪检测显示BMSCs高表达CD29(98.5%)、CD44(99.6%)和CD90(99.7%),而不表达CD34(4.0%)。对BAM作HE染色和扫描电镜显示未见明显的细胞残片。将TESG种植到网膜包裹两周后作HE检测显示：TEST表面有薄层上皮生长。动物实验中,直到取材时所有的动物均存活,术后2周切口都已痊愈,拔除尿管后未见明显尿道皮肤瘘发生。术后8周移植物和尿道之间没有明显的界限。术后2周,HE染色显示TESG完全被薄层上皮覆盖,TESG和正常组织的界限仍很清楚；到术后4周,TESG被覆上皮变厚,可见平滑肌生长。到第8周及16周,TESG有多层上皮覆盖。免疫组化显示抗AE1/AE3、抗uroplakinllla、和抗ZO-1均阳性,也进一步证实了上皮的再生情况。对照组中3只兔子在术后4周内均死亡,尸检显示尿道完全狭窄；对照组中另三只兔子排尿明显变细,显示有不同程度的尿道狭窄。 结论：利用BMSCs、SMCs和BAM构建TESG修复尿道缺损是可行的, BMSCs有可能促进尿路上皮的再生。
[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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