大鼠骨髓源性神经干细胞NCAM受体标记检测及超微结构实验研究

Experimental study on the detection of NCAM receptor and ultrastructure of rat BMSCs-D-NSCs

目的探讨神经细胞黏附分子(NCAM)受体作为大鼠骨髓源性干细胞检测标志,并利用原子力显微镜(AFM)扫描培养不同时段的细胞,获得细胞表面特异性超微结构。方法分离SD大鼠骨髓基质细胞,用神经干细胞培养基、脑源性神经细胞生长因子、碱性成纤维细胞生长因子、维甲酸持续诱导培养,在诱导培养后第6、8、10和12天应用SABC法免疫组化技术,检测NCAM受体表达情况;并对诱导培养后的细胞进行AFM扫描,检测细胞表面的形貌结构。结果光学显微镜下可见NCAM受体在诱导培养后第10天已经有表达。细胞超微结构的扫描测定显示细胞表面平均粗糙度在(11.2±1.5)nm,骨髓源性神经干细胞发育不同时段的细胞表面特征主要表现为细胞中央部表面粗糙而周边部光滑,细胞周边部均存在云雾状结构。结论NCAM受体可以作为大鼠骨髓源性神经干细胞的表面标记;AFM超微结构的明确,为进一步鉴定骨髓源性神经干细胞提供可能。

Objective To detect the feasibility of neural cell adhesion molecule （NCAM） receptor expression on the rat bone marrow stromal cells-derived neural stem cells （BMSCs-D-NSCs） as a cellular marker, and observe the specific ultrastructure of BMSCs-D-NSCs at different culture stages using atomic force microscopy （AFM）. Methods BMSCs were acquired from the bone marrow of rat lower limbs by puncture and then gradient centrifugation. NSCs culture medium, brain-derived neurotrophic factor （BDNF）, basic fibroblast growth factor （bFGF） and retinoic acid （RA） were used to induce the transformation from BMSCs into BMSCs-D-NSCs in vitro. The expression of NCAM on the developed BMSCs-D-NSCs was examined with immunohistochemistry at day 6, 8, 10 and 12 after culture. The ultrastructure of BMSCs-D-NSCs was then observed under AFM. Results The expression of NCAM receptor had been seen at day 10 after culture. AFM showed that surface average roughness of BMSCs-D-NSCs was （11.2±1.5） nm. The main features of the cell ultrastructure were that the central part was rougher than the periphery and that there were cloudiness structures around the cells. Conclusion NCAM receptor can be used as the cellar surface marker of BMSCs-D-NSCs. AFM can display precisely the ultrastructure of BMSCs-D-NSCs, which offer an access for further study of BMSCs-D-NSCs.