王远政,陈龙,张一,佘荣峰,戴涛,田晓滨
WANG Yuanzheng,CHEN Long,ZHANG Yi,SE Rongfeng,DAI Tao,TIAN Xiaobin

• 1:贵州省人民医院骨科

摘要(Abstract)：

目的:探讨一种从人尿液中分离培养尿源干细胞(hUSCs),并诱导分化为软骨细胞的方法。方法:通过离心贴壁法从健康成人尿液中分离、培养hUSCs,采用CCK-8法检测hUSCs的细胞增殖能力;成骨诱导3周时,使用茜素红染色检测hUSCs的成骨分化能力;成脂诱导2周时,使用油红O染色检测hUSCs成脂分化能力;甲苯胺蓝染色从细胞形态检测其成软骨分化能力,Aggrecan和CollagenⅡ免疫组组织化学染色检测其成软骨分化能力。结果:原代培养第7~10天时,可观察到细胞形态为短梭或多角形的典型h USC形态表现,第10~15天时hUSCs细胞呈成纤维细胞样的细长形改变;CCK-8结果显示,hUSCs符合典型的细胞增殖曲线规律;成骨诱导分化3周时,茜素红染色后可见红色钙结节;成脂诱导分化2周时,油红O染色后可见红色脂滴;成软骨诱导分化3周时,甲苯胺蓝染色阳性,提示细胞可分泌大量蛋白多糖;Aggrecan和CollagenⅡ结果均显示,hUSCs胞浆及周围可见大量棕色颗粒。结论:运用离心贴壁法获取的hUSCs具有良好的增殖特性和多项分化潜能,具有良好的软骨细胞分化能力。
Objective: To explore the approach of isolating and cultivating human urine-derived stem cells( hUSCs) so as to induce and differentiate chondrocyte from hUSCs. Methods: Primary hUSCs were isolated from fresh urine. Cell proliferation was evaluated using the Cell Counting Kit-8( CCK-8). 21 days after osteogenic induction,Alizarin Red S staining was utilized to detect calcified matrix deposition. 14 days after adipogenic induction,Oil Red O was utilized to detect the lipid accumulation. 21 days after chondrogenic induction,toluidine blue was used to show extracellular matrix sulfated proteoglycans. The expression of the chondrogenic-related marker( Aggrecan and Collagen II) was detected by immunohistochemistry( IHC). Results: After several passages of primitive culture of hUSCs,they turned to the fibroblast-like morphology. CCK-8 assay showed that the cells were initial in the stationary phase on the first 0-1 day. Then the cells entered into rapid growth phase from the 1 st to 3 rd day. Then the cells maintained slower growth from the 7 th day. Alizarin Red staining showed direct evidence for amorphous calcium mineral deposits. Oil Red O staining demonstrated the formation of lipid droplets in cytoplasm in hUSCs. 21 days after chondrogenic induction,toluidine blue staining showed direct evidence for extracellular matrix sulfated proteoglycans. The up-regulated expression of the chondrogenic-related markers( Aggrecan and Collagen II) were detected by IHC. Conclusions:hUSCs are a population of mesenchymal stem cells with better ability of possessing robust self-renew and multi-potential differentiation. hUSCs can be differentiated to chondrocyte in vitro.

关键词(KeyWords)： 成体干细胞,人,尿;诱导;成骨细胞;软骨;成脂分化;组织工程
adult stem cells,human,urine;induction;osteoblast;chondrocyte;adipogenic differentiation;tissue engineering

Abstract:

Keywords:

基金项目(Foundation): 贵州省高层次创新型人才千人计划资助项目[GZSYQCC(2015)002];; 贵州省人民医院博士基金[GZSYBS(2017)04]

作者(Author): 王远政,陈龙,张一,佘荣峰,戴涛,田晓滨
WANG Yuanzheng,CHEN Long,ZHANG Yi,SE Rongfeng,DAI Tao,TIAN Xiaobin

DOI: 10.19367/j.cnki.1000-2707.2018.07.007

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