骨髓间充质干细胞体外诱导分化与标记_医学论文.docVIP

骨髓间充质干细胞体外诱导分化与标记_医学论文 骨髓间充质干细胞体外诱导分化与标记_医学论文 作者：吴晓林,李冬民,马德茂,张晓田,黄石,宋天保 【关键词】 骨髓 【Abstract】 AIM: To study the differentiation characteristics and the optimal dosage and timing for bromodeoxyuridine (Brdu) labeling of rat bone marrowderived mesenchymal stem cells (MSCs) in vitro. METHODS: The rat marrow stem cells isolated by using Percoll (1.073 kg/L) were cultured and proliferated. The third passage cells were induced with dexamethasome, βglycerophosphate, ascorbic acid2phosphate, IGF1 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. 【Keywords】 MSCs induced differentiation osteoblast Brdu 【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1.073 kg/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21 d，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15 μmol/L的Brdu溶液标记MSCs，孵育12，24，48，72和96 h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. 10 μmol/L Brdu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72 h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10 μmol/L，最佳时间是72 h. 【关键词】 骨髓间充质干细胞；诱导分化；成骨细胞；5溴脱氧尿嘧啶核苷 0引言 骨髓间充质干细胞(mesenchymal stem cells，MSCs)是一类具有多向分化潜能的组织干细胞. 因MSCs取材方便、对机体的损伤小、具有较强的传代增殖能力和免疫耐受性等特点而受到越来越多的关注. 本实验的目的是利用MSCs具有分化潜能的特点，在体外诱导该细