尿源性干细胞在体外向尿路上皮细胞和平滑肌细胞的分化

Urine-derived stem cells differentiate into urothelial cells and smooth muscle cells in vitro

背景:成体干细胞是存在于已分化组织中的多能干细胞,尿源性干细胞为新发现的成体干细胞,具有取材方便、高度增殖能力、多向分化潜能等优点,在组织工程学领域受到重视。目的:研究人尿源性干细胞体外分离获取的方法,以及体外向尿路上皮细胞和平滑肌细胞分化的可行性。方法:收集健康成人尿液标本,通过分离、培养获取尿源性干细胞;应用CCK-8实验检测细胞增殖能力,并绘制细胞生长曲线;应用流式细胞仪检测细胞表型;体外应用专用培养基诱导尿源性干细胞向尿路上皮细胞和平滑肌细胞分化;应用荧光定量PCR、免疫组织化学染色、免疫荧光细胞染色和Western blot鉴定尿源性干细胞的分化能力。结果与结论:①从健康成人尿液中成功分离提取出尿源性干细胞,细胞具有高度增殖能力,生长曲线呈现S型;②尿源性干细胞高表达间充质干细胞表面标记物,CD29表达率为98.11%,CD90表达率为95.74%;③尿源性干细胞经体外诱导分化14 d后,可以表达尿路上皮细胞特异性基因Cytokeratin 7、Cytokeratin 20、UroplakinⅢ和平滑肌细胞特异性基因α-SMA和SM22;④结果表明,尿源性干细胞经过体外诱导可以向尿路上皮细胞和平滑肌细胞分化,能够为尿路组织修复重建提供理想的种子细胞来源。

BACKGROUND:Adult stem cells are pluripotent stem cells that exist in differentiated tissues.Urine-derived stem cells are newly discovered adult stem cells.They have attracted increasing attentions in the tissue engineering,due to its advantages of convenient sampling,highly proliferative ability and multidirectional differentiation potential.OBJECTIVE:To investigate the separation and extraction method of urine-derived stem cells and to investigate the feasibility of differentiation into urothelial cells and smooth muscle cells in vitro.METHODS:Urine specimens were collected from healthy adults,and urine-derived stem cells were obtained by isolation and culture in vitro.Cell counting kit-8 assay was used to detect cell proliferation and plot cell growth curve.Cell phenotype was detected by flow cytometry.The differentiation into urothelial and smooth muscle cells was induced by special medium respectively in vitro.The cell differentiation was detected by quantitative PCR,immunohistochemical staining,immunofluorescence cell staining and western blot assay.RESULTS AND CONCLUSION:Urine-derived stem cells were successfully isolated from the urine specimens of healthy adults.Urine-derived stem cells possessed high proliferation rate and the cell growth curve exhibited an S-shape.Urine-derived stem cells exhibited high expression of CD29(98.11%)and CD90(95.74%),both of which are mesenchymal stem cell surface markers.After 14 days of induction in vitro,urine-derived stem cells were able to express urothelial cell specific genes Cytokeratin 7,Cytokeratin 20,Uroplakin II and smooth muscle cell specific genesα-SMA and SM22.These results suggest that urine-derived stem cells can differentiate into urothelial cells and smooth muscle cells after induction in vitro and can be used as ideal seed cells for urinary tract tissue engineering.