依达拉奉体外定向诱导人间充质干细胞向神经元样细胞的分化

背景：依达拉奉作为新型氧自由基清除剂，一般用于抑制脂质过氧化反应，减轻脑水肿，保护神经细胞。 目的：观察依达拉奉体外定向诱导人骨髓间充质干细胞向神经元样细胞分化的可行性。 设计、时间及地点：细胞学体外观察，于2007-12/2008-09广东医学院附属医院中心实验室完成。 材料：骨髓来源于创伤所致闭合性股骨骨折的成年患者，由广东医学院附属医院骨科提供。依达拉奉由南京先声药业生产，批号P2007123144254453。 方法：无菌抽取的骨髓经肝素化后，采用密度梯度离心法及贴壁筛选法分离获得人骨髓间充质干细胞，传至第5代按1× 108 L-1接种于6孔板内，设立2组，依达拉奉组细胞达50%融合时用含碱性成纤维生长因子、胎牛血清的L-DMEM预诱导24 h，PBS洗涤后再用20 mg/L依达拉奉无血清L-DMEM诱导24 h；空白对照组始终用含体积分数为10%胎牛血清的L-DMEM培养，不加任何预诱导剂和诱导剂。 主要观察指标：诱导分化后细胞形态变化，SP法免疫细胞化学鉴定神经元烯醇化酶、巢蛋白、胶质纤维酸性蛋白及微管相关蛋白2的表达。 结果：体外诱导1 h后，依达拉奉组胞体收缩，2 h后形成较长突起，5 h后呈典型神经元样细胞；空白对照组细胞仍呈对称的梭形，无突起形成。免疫组化结果显示，诱导6 h后依达拉奉组神经元样细胞的胞体及部分突起呈棕黄色，强表达神经元烯醇化酶，弱表达胶质纤维酸性蛋白和巢蛋白，不表达微管相关蛋白2；空白对照组上述4种特异性抗原均呈阴性表达。 结论：人骨髓间充质干细胞经依达拉奉体外诱导后，所分化的细胞具有神经元表型，但还不够成熟，处于向成熟神经元分化的中间阶段。

Edaravone-induced differentiation of human bone marrow mesenchymal stem cells into neuron-like cells in vitro

BACKGROUND: Edaravone, a new oxygen free radical scavenger, is used to inhibit lipid peroxidatic reaction, to reduce brain edema, and to protect neural cells. OBJECTIVE: To explore the possibility of differentiation of human bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells by edaravone in vitro. DESIGN, TIME AND SETTING: The cytology in vitro study was performed at the Central Laboratory, Hospital Affiliated to Guangdong Medical College from December 2007 to September 2008. MATERIALS: Bone marrow was collected from adult patients with trauma-induced closed femoral fracture, and supplied by Department of Orthopaedics, Affiliated Hospital, Guangdong Medical College. Edaravone was produced by Xiansheng, Nanjing, China (number P2007123144254453). METHODS: Bone marrow was sterilely separated from human. After heparinization, human BMSCs were harvested using density gradient centrifugation and adherence method. At the fifth passage, BMSCs at 1×108/L were incubated in the 6-well plate and divided into 2 groups. BMSCs in the edaravone group were 50% confluent and incubated in L-DMEM containing basic fibroblast growth factor and fetal bovine serum for 24 hours. After washing in PBS, these BMSCs were incubated in serum-free L-DMEM containing 20 mg/L edaravone for 24 hours. BMSCs in the blank control group were incubated in L-DMEM, supplemented with 10% fetal bovine serum. MAIN OUTCOME MEASURES: After induction and differentiation, cell morphology was measured. Neuron specific enolase, nestin, glial fibrillary acidic protein and microtubule-associated protein-2 expression were assessed by immunocytochemistry (SP method). RESULTS: In the edaravone group, after 1 hour of in vitro induction, bodies were contracted; long processes appeared at 2 hours; typical neuron-like cells were detected at 5 hours. In the blank control group, BMSCs were symmetrical spindle-shaped, without processes. Immunohistochemistry results showed that bodies and some processes of neuron-like cells were dark yellow, and strongly expressed neuron specific enolase, weakly expressed nestin and glial fibrillary acidic protein, but did not express microtubule-associated protein-2. BMSCs in the blank control group were negative for neuron specific enolase, nestin, glial fibrillary acidic protein and microtubule-associated protein-2. CONCLUSION: Following in vitro induction of edaravone, human BMSCs can be differentiated into neuron-like cells, which are not mature and in the middle stage of differentiation.