果糖和二硫苏糖醇对骨髓间充质干细胞冻存后活性及多能性的影响

摘要

背景：冻存是保证干细胞临床应用的关键步骤之一，但现有冻存技术常导致细胞活性降低、多能性丧失及分化能力下降。　　目的：探究果糖及二硫苏糖醇是否有助于维持冻存后骨髓间充质干细胞多能性及成骨分化潜能。　　方法：分离培养SD大鼠骨髓间充质干细胞，在细胞冻存前分别用果糖(200μmol/L)，二硫苏糖醇(500μmol/L)及果糖(200μmol/L)+二硫苏糖醇(500μmol/L)预处理1 h。冻存6个月后，复苏细胞并用倒置显微镜观察细胞形态，MTT实验检测细胞活性，定量PCR检测相关干性基因(Nanog，Oct4及Sox2)的表达，碱性磷酸酶活性测试及茜素红染色检测复苏骨髓间充质干细胞成骨分化能力。　　结果与结论：①复苏后各组细胞在形态上无明显差别；②果糖预处理及联合预处理有助于骨髓间充质干细胞活性维持；③二硫苏糖醇预处理可显著促进骨髓间充质干细胞多能性相关基因Nanog及Sox2的表达；④果糖、二硫苏糖醇及联合预处理皆有助于维持骨髓间充质干细胞成骨分化潜能，但以二硫苏糖醇及联合预处理组效果最佳；⑤结果表明，果糖预处理有助于维持冻存骨髓间充质干细胞活性，二硫苏糖醇有助于维持冻存骨髓间充质干细胞多能性及成骨分化能力。%BACKGROUND:Cel cryopreservation is required for clinical use of stem cel s, and the current process of cryopreservation however may be harmful to cel viability, pluripotency and differentiation capacity. OBJECTIVE:To explore the effect of fructose and dithiothreitol on pluripotency and osteogenesis of cryopreserved bone marrow mesenchymal stem cel s. METHODS:Bone marrow mesenchymal stem cel s were isolated from the bone marrow of Sprague-Dawley rats and pretreated with fructose (200μmol/L), dithiothreitol (500μmol/L) or combined components before cryopreservation. Then the cel s were cryopreseved for 6 months and the morphology of cel s was observed by inverted microscopy. The cel viability was evaluated by MTT, and real-time PCR was used to detect the mRNA expression of Nanog, OCT4 and Sox2. Alkaline phophatase activity assay and alizarin red staining were utilized to detect the osteogenic capacity of bone marrow mesenchymal stem cel s. RESULTS AND CONCLUSION:Images captured by inverted microscopy showed no significant difference in cel morphology between groups. The MTT results indicated that fructose and combined pretreatment could promote the cel viability of bone marrow mesenchymal stem cel s after cryopreservation, while the real-time PCR results demonstrated that dithiothreitol significantly facilitated the expression of Naogo and Sox2 in bone marrow mesenchymal stem cel s. Moreover, ALP activity assay and alizarin red staining confirmed the positive effects of fructose, dithiothreitol and combined pretreatment on osteogenic capacity of bone marrow mesenchymal stem cel s after cryopreservation, and the best effects were found after pretreatment with dithiothreitol and combined components. Overal , these findings indicate that fructose pretreatment is beneficial for cel viability of cryopreseved bone marrow mesenchymal stem cel s, and dithiothreitol contributes to maintaining the pluripotency and osteogenesis capacity of cryopreseved bone marrow mesenchymal stem cel s.