共培养法神经细胞诱导骨髓间充质干细胞向神经元的分化

Differentiation of bone marrow mesenchymal stem cells into neurons induced by neural cells by co-culture method

背景：目前神经细胞是否具有直接诱导骨髓间充质干细胞分化为神经元的作用还未见报道。目的：拟在体外建立入骨髓间充质干细胞和神经细胞的共培养体系，观察共培养条件下神经细胞对骨髓间充质干细胞分化为神经元的影响。设计、时间及地点：分组对照，体外细胞学实验，于2006—12／2007—12在山东省青岛大学医学院附属医院中心实验室完成。材料：骨髓间充质干细胞来源于青岛大学医学院附属医院行自体干细胞移植治疗的糖尿病患者骨髓血，神经细胞取材于分娩过程中窒息死亡的新生儿脑组织，传至第3代用于实验。用于细胞共培养的Transwell双层培养皿为Corning Costar公司产品，培养体系孔径小于3.0μm．细胞不会迁徙通过．但上、下层培养液可互通融合。方法：共培养组将神经细胞按1×10^6密度接种于Transwell双层培养皿的底层，上层接种骨髓间充质干细胞，加入LG-DMEM培养基,培养4-5d。对照组上、下层均接种骨髓间充质干细胞。主要观察指标：观察骨髓间充质干细胞的形态变化，免疫荧光染色检测骨髓间充质干细胞中神经元特异性标志物的表达。结果：共培养组骨髓间充质干细胞生长伸展，呈放射状突起．并可相互连接。神经元特异性烯醇化酶阳性率为（32．7±11．5）％．表现神经元细胞的特性。对照组骨髓间充质干细胞形状扁而宽，未形成神经样形态结构，神经元特异性烯醇化酶呈阴性。结论：神经细胞生长过程中提供的微环境对骨髓间充质干细胞分化为神经元具有诱导促进作用。

BACKGROUND： There are no studies on differentiation of bone marrow mesenchymal stem cells （BMSCs） into neurons directly induced by neural cells. OBJECTIVE： To establish the co-culture system between BMSCs and neural cells in vitro, and to study the influence of neural cells on the differentiation of BMSCs into neuron in the co-culture system. DESIGN, TIME AND SETTING： The in vitro cytology control experiment was performed at the Central Laboratory of Hospital Affiliated to Medical College of Qingdao University fi＇om December 2006 to December 2007. MATERIALS： BMSCS were harvested from bone marrow of patients with diabetes meliuts, who underwent autologous stem cell transplantation at the Hospital Affiliated to Medical College of Qingdao University. Neural cells were collected from brain tissue of infants die of asphyxia during delivery. The third passage of neural cells was used in this study. Transwell double〈leck culture dish was purchased from Coming Costar, with a pore diameter of less than 3.0 μ m. Cells could not traverse, but the medium could traverse. METHODS： Neural cells were incubated in Transwell double-deck culture dish at a density of 1 × 10^6 in the co-culture group. BMSCs were incubated in the upper layer in LG-DMEM medium for 4-5 days. BMSCs were incubated in both layers in the control group. MAIN OUTCOME MEASURES： The morphological changes of BMSCs were observed and the special markers of neurons cells in BMSCs were examined by immunofluorescence. RESULTS： BMSCs in the co-culture group grew slowly, showing radial processes and connected each other. Positive rate of neuron specific enolase was （32.7 ± 11.5）%. BMSCs in the control group were flat and wide, and negative for neuron specific enolase. CONCLUSION： Microenvironment provided by neural cells promotes the differentiation of BMSCs into neurons.