摘要
目的:探讨还原型谷胱甘肽(reduced glutathione,GSH)对人脐带间充质干细胞(humanumbilical cord mesenchymal stem cells,hUC-MSCs)的成软骨诱导是否有促进作用。方法:分离培养人脐带间充质干细胞并鉴定其特异性。在生长因子(tansforming growth factor beta 1,TGF-β1)存在的条件下,用500μmol/L GSH诱导hUC-MSCs向软骨样细胞分化。倒置相差显微镜观察诱导前后细胞的形态变化;甲苯胺蓝染色及免疫荧光反应分别检测Ⅱ型胶原(COL2A1)、葡萄糖胺聚糖(GAG)(蛋白水平定性);阿利新蓝法及羟脯氨酸法分别检测COL2A1、GAG含量(蛋白水平定量);qPCR在分子水平检测COL2A1、GAG mRNA表达,观察诱导前后细胞的基质分泌情况。结果:GSH连续诱导培养21d后,基本诱导培养基组(B组)、基本诱导培养基+0.5%二甲基亚砜(dimethylsulfoxide,DMSO)组(BD组)及基本诱导培养基+0.5%DMSO+500μmol/L GSH组(BDG组)细胞甲苯胺蓝染色及免疫荧光反应均呈阳性;BDG组GAG和COL2A1含量及mRNA表达量均要高于B组、BD组,具有显著的统计学差异(P<0.05)。结论:GSH可促进hUC-MSCs向软骨样细胞诱导分化。
Objective： Reduced glutathione （GSH） were discussed whether GSH could promote the chondrogenic induction ability of human umbilical cord mesenchymal stem cells （hUC-MSCs）. Methods： hUC-MSCs were isolated from human umbilical cord and their specificity was identified, then, induced into cartilage-like cells in chondrogenic induction medium with tansforming growth factor beta 1 （TGF-β1）, especially with GSH. The morphological change before and after induction was observed through inverted phase contrast microscope, Type Ⅱcollagen（COL2A1） and glycosaminoglycan （GAG） were analyzed qualitatively by toluidine blue and immunofluorescence technique, respectively, the contents of COL2A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2A1 were assayed by real-time fluorescence quantitative PCR. Results： After continuously cultured for 21days with GSH, Toluidine blue staining and immunofluorescence reaction were all positive in basic induction medium group （group B）, basic induction medium＋0.5% dimethylsulfoxide （DMSO） group （group BD） and basic induction medium＋0.5% DMSO＋500μmol/L GSH group （group BDG）. Moreover, compared with in group B and group BD, the content of COL2A1 and GAG in group BDG relatively increased and the mRNA expression level of COL2A1 and GAG also comparatively increased（P＜0.05）, and both had a significant statistical significance（P＜0.05）. Conclusion： GSH might promote the chondrogenic differentiation of hUC-MSCs.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第3期1-8,共8页
China Biotechnology
基金
广东省自然科学基金资助项目(9151008901000050)
关键词
还原型谷胱甘肽
人脐带间充质干细胞
软骨样细胞
蛋白多糖
Ⅱ型胶原
Reduced glutathione
Human umbilical cord mesenchymal stem cells
Chondrogenic differentiation
Dlycosaminoglycan
Type Ⅱcollagen
