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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: To investigate whether reduced glutathione (GSH) can promote chondrogenic induction of human umbilical cord mesenchymal stem cells (hUC-MSCs). Methods: Human umbilical cord mesenchymal stem cells were isolated and identified and their specificity was identified. HUC-MSCs were induced to differentiate into chondrocyte-like cells in the presence of 500 μmol / L GSH in the presence of tansforming growth factor beta 1 (TGF-β1). The morphological changes of cells before and after induction were observed by inverted phase contrast microscope. COL1A and GAG were detected by toluidine blue staining and immunofluorescence reaction. Aliskin blue and hydroxyproline Methods COL2A1 and GAG contents (protein level quantitation) were detected by qPCR method. The expression of COL2A1 and GAG mRNA was detected by qPCR at the molecular level. The secretion of matrix was observed before and after induction. RESULTS: After induction of GSH for 21 days, basal induction medium group (group B), basic induction medium + DMSO group (0.5%) and BD group (basic induction medium + 0.5% DMSO Toluidine blue staining and immunofluorescence reaction in + 500μmol / L GSH group (BDG group) were all positive. The content and mRNA expression of GAG and COL2A1 in BDG group were significantly higher than those in group B and BD P <0.05). Conclusion: GSH can promote hUC-MSCs to differentiate into chondrocyte-like cells.