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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 investigate the expression of neural cell adhesion molecule (NCAM) receptor as a marker of rat bone marrow-derived stem cells and to observe the cell surface-specific ultrastructure by scanning the cells at different time points with atomic force microscopy (AFM). Methods SD rat bone marrow stromal cells were isolated and cultured with neural stem cell culture medium, brain-derived neurocyte growth factor, basic fibroblast growth factor and retinoic acid. After 6, 8, 10 and 12 days SABC immunohistochemical technique was used to detect the expression of NCAM receptor. AFM scanning of cultured cells was performed to detect the morphology of cell surface. Results Under light microscope, NCAM receptor was expressed on the 10th day after induction culture. Scanning of cell ultrastructure showed that cell surface roughness was (11.2 ± 1.5) nm in average. The cell surface features at different time points of bone marrow-derived neural stem cell development were mainly characterized by a rough surface at the central part of cell and a smooth peripheral part, There is cloud-like structure. Conclusion The NCAM receptor can be used as the surface marker of rat bone marrow-derived neural stem cells. The ultrastructure of AFM is clear, which provides a possibility for further identification of bone marrow-derived neural stem cells.