摘要
目的探讨间接共培养法诱导人脂肪间充质干细胞向尿路上皮细胞定向分化的可行性。方法人脂肪间充质干细胞提取自吸脂术患者的废弃脂肪组织中，经流式细胞法来鉴定人脂肪间充质干细胞的表型。应用Transwell培养系统构建间接共培养模型，上层种植输尿管上皮细胞（1×104／cm2），下层种植脂肪间充质干细胞（0．5×10^4／cm2），共培养14d。采用免疫荧光方法鉴定尿路上皮标志物[细胞角蛋白-18（CK-18），尿斑蛋白2（UP2）]表达。将诱导后的细胞种植于I型胶原＋聚乳酸混合电纺丝输尿管支架材料上，体外培养7d，免疫荧光法鉴定诱导后的细胞在输尿管支架材料上尿路上皮标志物表达。苏木素-伊红（HE）染色、Livedead法检测观察诱导后细胞生长。结果共培养7d后，诱导后的细胞形态呈现尿路上皮细胞样，14d后，免疫荧光检测表明，40％～50％的干细胞表达尿路上皮标志物（CK-18及UP2）。诱导后的细胞种植在输尿管支架材料上，细胞增殖生长良好，呈铺展状态生长，活细胞占总数的90％以上（Livedead），并且表达尿路上皮标志物。结论使用间接共培养法能成功诱导人脂肪间充质干细胞向尿路上皮细胞分化，为输尿管等泌尿系统组织工程研究提供了一种新的种子细胞。
Objective To evaluate the transdifferentiation potential of human adipose-derived mesenchymal stem cells into urothelial cells by indirect co-cuhure. Methods Human adipose-derived mes- enchymal stem cells were isolated from the waste tissue of liposuction and characterized by flow cytometry. The indirect coculture model was constructed by Transwell culture system： the ureter urothelial cells were seeded in the upper chamber （ 1 x 104/cm2 ） , and the adipose-derived stem cells were seeded in the lower chamber （0. 5×10^4/cm^2）. After the cells were co-cultured for 14 days, immunofluorescence assays were done to evaluate the expression of urothelial lineage markers [ Cytokeratin-18 （CK-18, Uroplakin 2 （UP2） ] by the stem cells. The induced stem cells were then seeded onto the collagen I/poly-lactic acid electrospinning ureteral scaffold and cuhured for 7 days. Immunofluorescence assays were performed to e- valuate urothelial markers in the scaffold by the induced cells, and hematoxylin and cosin （HE） and live- dead stainings were done to evaluate the cell growth. Results After co-culture for 7 days, the induced cells changed their morphology into urothelial-like shape, and 14 days later the induced cells expressed the markers （CK-18, UP2） of urothelial cells. The induced cells grew well on the collagen L/poly-lactic acid electro-spinning scaffold, spread on the scaffold and more than 90% cells were alive on the scaffold （livedead）. In addition, the induced cells expressed the urothelial lineage markers on the scaffold in vitro. Conclusion Human adipose-derived mesenchymal stem cells could be induced into urothelial lineage differentiation by indirect co-culture, providing a new cell resource for ureter and other urinary tract tissue engineering.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第5期927-929,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81070555）
北京市自然科学基金资助项目（2092029）
军队临床高新技术重点项目（413DG63J）
关键词
脂肪间充质干细胞
尿路上皮
共培养
Adipose-derived mesenehymal stem cells
Urothelial cells
Coculture
