兔骨髓间充质干细胞向尿路上皮分化后构建膀胱组织工程化移植物

背景：尿路上皮细胞是泌尿系组织工程领域重要的种子细胞，但是难以体外大量扩增；有研究表明骨髓间充质干细胞可以向尿路上皮细胞分化，但关于分化后细胞在植入动物体内后上皮生成情况，以及分化后细胞在组织工程领域的具体应用研究尚不多。 目的：分离、扩增兔骨髓间充质干细胞，诱导骨髓间充质干细胞向尿路上皮细胞分化并与兔膀胱脱细胞基质构建组织工程化移植物，了解分化后细胞作为种子细胞的效果。 方法：12只8周龄雄性新西兰大白兔胫骨穿刺，抽取骨髓，密度梯度离心法分离骨髓间充质干细胞，第4或5代细胞以条件培养基培养2周，进行分化后细胞的鉴定。随后将分化后细胞种植在膀胱脱细胞基质上构建组织工程化移植物，进行膀胱修补；另12只动物作为对照组以尿路上皮细胞与膀胱脱细胞基质构建复合物行膀胱修补。 结果与结论：骨髓间充质干细胞培养成功并在体外扩增，由条件培养基培养诱导分化后，PCR检测提示分化后细胞干细胞标志物CD44表达降低，而上皮细胞标志物UP1a表达升高，行免疫荧光检测发现分化后细胞能表达尿路上皮特异性标志物UP1a，而骨髓间充质干细胞无表达。分化后细胞构建的组织工程化移植物在膀胱修补2周后可形成稳定连续的上皮覆盖，上皮层厚度与尿路上皮细胞构建的组织工程化移植物类似。提示骨髓间充质干细胞向尿路上皮诱导分化后可作为泌尿系组织工程的种子细胞，可能是尿路上皮细胞之外的又一新选择。

Construction of bladder tissue-engineered grafts by urothelium-induced bone marrow mesenchymal stem cells and bladder acellular matrix

BACKGROUND:Urothelial cells are important seeding cells for urinary tissue engineering, but they are difficult to proliferate in vitro. Several studies have shown that bone marrow mesenchymal stem cells can differentiate into urothelial cells, but how these cells functions in vivo in epithelium generation after implantation, and the application of these cells in tissue engineering, are rarely studied. OBJECTIVE: To explore the isolation and proliferation of rabbit bone marrow mesenchymal stem cells that are induced into urothelial cells in combination with rabbit bladder acellular matrix to construct tissue-engineered grafts, and to assess the effect of the induced cells as seeding cells. METHODS: Twelve 8-week-old male New Zealand white rabbits were chosen to obtain bone marrow samples through tibia puncture, and to isolate bone marrow mesenchymal stem cells by density gradient centrifugation. Then the fourth or fifth generation of bone marrow mesenchymal stem cells were cultured in conditioned medium for 2 weeks, and then identified by PCR and immunofluorescence. After that, the induced cells were seeded on rabbit bladder acellular matrix to construct tissue-engineered grafts for bladder repairing. Another 12 rabbits  served as control group, and urothelial cells combined with bladder acellular matrix was used for bladder repairing. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells were successfully cultured and proliferated in vitro. After induction, PCR detection suggested that stem cell marker (CD44) expression decreased, and epithelial cell marker (UP1a) expression increased in the induced cells. Immunofluorescence staining demonstrated that the induced cells rather than bone marrow mesenchymal stem cells were positive for specific urothelial marker, UP1a. A stable continuous epithelial layer was observed on tissue-engineered grafts constructed by induced cells after 2 weeks, similar to the grafts built by urothelial cells. Induced bone marrow mesenchymal stem cells can differentiate into urothelial cells that can be used as seeding cells for urinary tissue engineering, which may be another choice out of urothelial cells.