脂肪干细胞复合膀胱脱细胞基质促进膀胱再生的实验研究

Bladder reconstruction with seeding adipose-derived stem cells onto bladder acellular matrix grafts

目的探讨脂肪干细胞（adipose—derived stem cell，ADSC）在兔的组织工程膀胱重建中的应用。方法采用自体脂肪干细胞和膀胱脱细胞基质（bladder acellular matrix graft，BAMG）构建兔组织工程膀胱。应用酶消化法分离培养兔自体的ADSC，并进行流式细胞鉴定。对新鲜的兔膀胱组织进行脱细胞处理制得BAMG。将ADSC扩增后接种于BAMG表面并进行体外培养，获得组织工程膀胱。对24只雄性新西兰兔行40％～50％膀胱部分切除，分为实验组和对照组，每组12只。实验组采用自体培养的ADSC构建的组织工程膀胱对缺损膀胱进行修复，对照组采用单纯的BAMG进行修复。术后24周进行膀胱造影并取材观察组织修复再生情况。结果镜下观察ADSC贴壁后绝大多数为纺锤形，细胞之间以细丝拉网。流式细胞仪检测结果CD90、CD44、CD105、CD166、CD34表达均为阳性，CD106、CD45表达为阴性。ADSC种植于BAMG表面生长良好。修补24周后，对照组和实验组膀胱容量分别为术前的（69．33±5．05）％和（94．68±3．31）％。组织染色分析对照组显示上皮层正常，黏膜下纤维组织增生而肌层较薄；实验组则显示了与自体膀胱相似的正常的3层组织结构。结论采用ADSC和BAMG构建组织工程膀胱是理想的膀胱替代修补材料。

Objective To assess the feasibility of seeding adipose-derived stem cells （ADSCs） onto bladder acellular matrix grafts （BAMGs） for bladder reconstruction in a rabbit model. Methods Autologous ADSCs were isolated, expanded and identified by flow cytometry. In the experimental group, ADSCs were seeded onto BAMGS for reconstructing bladder defects in 12 male rabbits. Unseeded BAMGs were used for bladder reconstruction in the control group of 12 rabbits. Cystography was performed at 24 weeks after grafts implantation. Following cystography, the animals were scarified and grafts were harvested; H＆E and immunohistochemical staining were performed with cytokeratin AE1/AE3, smooth muscle α-actin and S-100 markers. Results Flow cytometry demonstrated that the ADSCs expressed CD90, CD44, CD105 , CD166 and CD34, but not CD45 or CD106. The cells demonstrated good biocompatibility with BAMGs. At 24 weeks, in the experimental group, the reconstructed bladders reached a mean volume of （94.68 ± 3.31 ） % of the precystectomy bladder capacity. Complete regeneration of smooth muscle and nerve tissue was evident. Regenerated SMCs, urothelium and nerve cells stained positively for a-smooth muscle actin, AE1/AE3 and S- 100. In the control group, the mean bladder volume was （69.33 ~ 5.05 ）% of the pre-cystectomy volume. Histologically, the control group was characterized by multi-layered urothelium without evidence for organized muscle or nerve tissue. Conclusion The tissue engineering bladder constructed by ADSCs and BAMG can be used as an ideal biomaterial to replace and repair the bladder.