【摘要】 目的:探讨骨髓间充质干细胞(MSCs)在体外的诱导分化及5-溴脱氧尿嘧啶核苷(Brdu)标记MSCs的可行性.方法:利用密度为1.073kg/L的percoll分离骨髓的单个核细胞,体外培养与扩增MSCs;取生长良好的第3代细胞,用成骨细胞分化培养液培养21d,Ⅰ型胶原免疫组化染色和碱性磷酸酶(AKP)组化染色;分别以浓度为5,10和15μmol/L的Brdu溶液标记MSCs,孵育12,24,48,72和96h后免疫组化检测Brdu,确定最佳标记量和最佳标记时间.结果:诱导分化第21日,AKP染色呈强阳色,Ⅰ型胶原免疫染色呈阳性.10μmol/LBrdu标记MSCs的效果最好,随着孵育时间的延长,Brdu标记率逐渐增高,标记72h后标记率在90%以上.结论:MSCs在体外一定条件下可定向诱导分化为成骨细胞,用Brdu标记MSCs的最佳浓度为10μmol/L,最佳时间是72h.更多还原

【Abstract】 AIM: To study the differentiation characteristics and the optimal dosage and timing for bromodeoxyuridine (Brdu) labeling of rat bone marrow-derived mesenchymal stem cells (MSCs) in vitro. METHODS: The rat marrow stem cells iso- lated by using Percoll (1.073 kg/L) were cultured and proliferated. The third passage cells were induced with dexamethasome, β-glycerophosphate, ascorbic acid-2-phosphate, IGF-1 and insulin. On day 21, the cells were analyzed by immunohistochemistry for type Ⅰ collagen and by histochemistry for alkaline phosphatase (AKP). The purified MSCs were incubated with Brdu at different concentrations (5, 10 and 15 μmol/L) for different time periods (12, 24, 48, 72 and 96 h), followed by immunohistochemistry for Brdu to identify the optimal Brdu concentration and incubation time period. RESULTS: After incubation for 21 d for induced differentiation into osteoblasts, type Ⅰ collagen and AKP were positively expressed. The incubation with 10 μmol/L Brdu for 72 h achieved over 98% of the labeling rate without producing obvious cell damages. CONCLUSION: MSCs can be induced to differentiate into osteoblasts under certain conditions in vitro. The optimal Brdu labeling concentration and time period are 10 μmol/L and 72 h, respectively.更多还原