人尿源性干细胞的分离培养及诱导分化为平滑肌细胞  

Isolation,culture and differentiation of human urine-derived stem cells into smooth muscle cells

作　　者：陈佳汇 戴晓琪 徐彦钢 李远超 黄妹 詹一飞 杜宇轩 李鎏强 郭耀川 卞军 赖德辉[1] Chen Jiahui;Dai Xiaoqi;Xu Yangang;Li Yuanchao;Huang Mei;Zhan Yifei;Du Yuxuan;Li Liuqiang;Guo Yaochuan;Bian Jun;Lai Dehui(Department of Urology,Fifth Affiliated Hospital of Guangzhou Medical University,Guangzhou 510000,Guangdong Province,China;Department of Urology,Guangzhou Integrated Traditional Chinese and Western Medicine Hospital,Guangzhou 510000,Guangdong Province,China;Clinical Acupuncture and Moxibustion Rehabilitation College,Guangzhou University of Chinese Medicine,Guangzhou 510000,Guangdong Province,China)

机构地区：[1]广州医科大学附属第五医院泌尿外科,广东省广州市510000 [2]广州市中西医结合医院泌尿外科,广东省广州市510000 [3]广州中医药大学临床针灸康复学院,广东省广州市510000

出　　处：《中国组织工程研究》2025年第19期4076-4082,共7页Chinese Journal of Tissue Engineering Research

基　　金：广州市科技计划项目(202201010834),项目负责人:卞军;广东省自然科学基金-面上项目(2021A1515010065),项目负责人:卞军;广州市卫生健康科技项目(20211A011103),项目负责人:卞军;2022年教育部产学合作协同育人项目(220904082210823),项目负责人:赖德辉;2022年广州医科大学学生创新能力提升计划项目(106),项目负责人:赖德辉;广东省自然科学基金博士启动项目(2017A030310148),项目负责人:赖德辉。

摘　　要：背景:传统尿路修复重建手段受限于供体短缺、并发症多以及生理功能恢复不理想等问题,组织工程策略为此领域提供了新方向。鉴于尿路主要由肌性组织构成,其中关键在于发掘适合的种子细胞并高效诱导分化为平滑肌细胞,但关于不同平滑肌细胞诱导方案效能的对比研究仍较为匮乏。目的:旨在分离、培养及鉴定人尿源性干细胞,并比较两种不同成平滑肌诱导方案的效果。方法:采用多次离心法从11份健康成人志愿者尿液中分离提取尿源性干细胞,使用流式细胞仪进行表面标志物的鉴定,通过成骨、成脂诱导分化来验证尿源性干细胞的多向分化潜能。尿源性干细胞分别在含转化生长因子β1以及转化生长因子β1联合血小板衍生生长因子的成平滑肌细胞诱导分化培养基中诱导分化14 d,采用免疫荧光染色和Western blot检测平滑肌特异性蛋白(α-SMA、SM22)的表达差异。结果与结论:①成功从8份健康人尿液中分离出尿源性干细胞,细胞呈“米粒”样,具有很好的分裂增殖能力;②尿源性干细胞高表达间充质干细胞表面标志物CD73、CD90和CD44,极低表达造血干细胞表面标志物CD34和CD45,不表达CD19、CD105和HLA-DR;③经成骨和成脂诱导分化后,可见明显的钙结节和脂滴形成,茜素红染色和油红O染色结果呈阳性;④成平滑肌诱导培养14 d,免疫荧光染色显示转化生长因子β1/血小板衍生生长因子组尿源性干细胞成平滑肌诱导分化率显著高于转化生长因子β1组(P<0.005);⑤成平滑肌诱导培养14 d,Western blot检测显示转化生长因子β1/血小板衍生生长因子组α-SMA和SM22蛋白表达量显著高于转化生长因子β1组(P<0.005)。结果表明:尿源性干细胞可以通过多次离心法无创分离获取;相较于单纯转化生长因子β1,转化生长因子β1/血小板衍生生长因子联合应用能显著提高尿源性干细胞诱导分化为平滑肌细胞�BACKGROUND:Traditional methods of urinary tract reconstruction are limited by donor scarcity,high complication rates,and suboptimal functional recovery.Tissue engineering strategies offer new directions in this field.Since the urinary tract is mainly composed of muscle tissue,the key is to find suitable seed cells and efficiently induce them to differentiate into smooth muscle cells.Comparative studies on the efficacy of different smooth muscle cell induction regimens are still lacking.OBJECTIVE:To isolate,culture,and identify human urine-derived stem cells,and to compare the effects of two different induction protocols.METHODS:Human urine-derived stem cells were isolated from urine samples of 11 healthy adult volunteers by multiple centrifugations.Surface markers were identified by flow cytometry.The multi-directional differentiation potential of human urine-derived stem cells was verified through osteogenic and adipogenic differentiation.Differentiation was induced by transforming growth factor-β1 or transforming growth factor-β1 combined with platelet derived growth factor for 14 days.Immunofluorescence staining and western blot assay were employed to compare the expression differences of smooth muscle-specific proteins(α-SMA and SM22).RESULTS AND CONCLUSION:(1)Urine-derived stem cells were successfully isolated from the eight urine samples of healthy people.These cells exhibit a“rice grain”-like morphology and possess a robust proliferative capacity.(2)Urine-derived stem cells exhibited high expression of mesenchymal stem cell surface markers(CD73,CD90,and CD44)and extremely low expression of hematopoietic stem cell surface markers(CD34 and CD45).These cells did not express CD19,CD105,and HLA-DR.(3)After osteogenic and adipogenic differentiation,the formation of calcium nodules and lipid droplets was observed,with positive staining results from Alizarin Red S and Oil Red O staining.(4)After 14 days of smooth muscle induction culture,immunofluorescence staining revealed that the smooth muscle differenti

关 键 词：尿源性干细胞 诱导分化 平滑肌细胞 转化生长因子β1 血小板衍生生长因子 组织工程 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R69