新西兰兔骨髓基质细胞构建组织工程膀胱的实验研究

Marrow stromal cells application: Reconstruction of the bladder of New zealand white rabbits by tissue engineering techniques

目的 :研究骨髓基质细胞 (MSCs)在兔组织工程膀胱中的应用 .方法 :按文献改进方法制作膀胱无细胞基质(BAMG) ,从兔胫骨中分离出MSCs,体外扩增后接种于BAMG外表面 ,在低糖DMEM (LG DMEM )培养 12h ,与BAMG复合成组织工程膀胱 ,对同一兔实施膀胱次全切术 ,用组织工程膀胱替代 .2 0只兔实施了保留三角区的膀胱次全切术 .实验随机分为 3组 .A组 6只直接缝闭三角区 ;B组 7只用膀胱形状的BAMG进行重建 ;C组 7只用复合自体MSCs的组织工程膀胱进行替代手术 .术前术后分别记录膀胱测压和放射线造影资料 .12wk时无痛处死动物 ,进行大体解剖和组织染色分析 .结果 :体外扩增时间平均为 2 1d .A ,B和C组膀胱容积分别为术前的 2 2 % ,85 %和 95 % ,顺应性分别为术前的 11% ,10 4 %和 10 7% .大体解剖显示A组仅有很小的储水能力 ,而B ,C组具有正常膀胱形态 ,组织染色分析A ,B组显示上皮层正常 ,粘膜下纤维组织增生而肌层较薄 ;C组则显示了与自体膀胱相似的正常的 3层组织结构 .结论 :膀胱次全切除后原位缝合膀胱容积和顺应性均不能恢复 ,单纯应用BAMG膀胱容积和顺应性能够恢复正常 ,但逼尿肌层较薄 ,应用BAMG复合自体ASCs或MSCs的组织工程膀胱替代 ,术后 12wk膀胱容积和顺应性能够恢复正常 。

AIM: To investigate the function parameters of tissue engineered bladders with marrow stromal cells(MSCs). METHODS: The MSCs and the bladder-shaped accellular organ specific matrix (BAMG) were obtained from the New Zealand White rabbits. The exterior surface of BAMG was seeded with the resuspended MSCs. A total of 20 rabbits underwent a trigone-sparing cystectomy. The animals were randomly assigned to three groups. Group A ( n =6) underwent a closure of the trigone without a reconstructive procedure. Group B ( n =7) underwent a reconstruction using a BAMG only and Group C ( n =7) underwent a reconstruction using a BAMG plus autologous MSCs. Preoperative and postoperative cystometry and radiography studies were performed serially. Animals were euthanized at 12 weeks. Gross and histological analyses were also performed. RESULTS: The average time elapsed between the initial marrow biopsy and the final implantation of the tissue-engineered neo bladders was (21±3) days ( x±s ). Post operation, the average capacities in Group A, B and C were 22%, 85% and 95%, respectively and showed a marked difference in bladder compliance (11%, 104%, and 107% respectively). Histologically, group C showed a normal cellular organization consisting of a trilayer of urothelium, submucosa and muscle. The Group A, B presented a pattern of normal urothelial cells with a thickened fiberotic submucosa and a thin layer of muscle fibers. CONCLUSION: Our study demonstrates that the MSCs can be successfully used in rabbits bladder augmentations and replacement by tissue engineering techniques.