海马中56kD蛋白诱导人神经干细胞向神经元分化的作用

在已证明切割海马伞海马中差异表达的56kD蛋白具有诱导神经干细胞迁移作用的基础上,进一步探讨其诱导神经干细胞向神经元和AChE阳性神经元分化的作用。取切割SD大鼠海马伞后14d及正常海马组织的匀浆进行非变性聚丙烯酰胺凝胶电泳(Native-PAGE)。将用无血清培养技术获取的人胚神经干细胞球分别与切割海马伞后14d和正常海马56kD差异蛋白的胶条及空白对照胶条共培养,在倒置显微镜下观察神经干细胞球中细胞的迁移和分化情况,于第21d分别应用MAP-2免疫荧光和AChE组织化学方法检测人神经干细胞分化为神经元和AChE阳性神经元的情况,并进行神经元的计数、细胞面积、细胞周长的图像处理和统计学分析。结果显示,分化的MAP-2阳性神经元的数量、细胞面积及细胞周长三项指标切割组最好,正常组次之,而空白对照组最差;三组AChE组织化学染色均为阴性结果。上述结果提示切割海马伞后海马中差异表达的56kD蛋白具有诱导神经干细胞向MAP-2阳性神经元分化的作用,但不能诱导神经干细胞向AChE阳性神经元分化。

EFFECT OF THE 56 kD PROTEIN IN HIPPOCAMPUS ON INDUCING HUMAN NEURAL STEM CELLS DIFFERENTIATING INTO NEURONS

Based on the evidence that the differential 56 kD protein in the fimbria-transected hippocampus had effect on inducing human neural stem cells (hNSCs) migrating, effects of the 56 kD protein inducing hNSCs differentiating into neurons and AChE-positive neurons were further studied. The hippocampi of SD rats on the 14th day after fimbria transection and the normal ones were extracted for Native-PAGE. The spheres of hNSCs gained by the serum-free culture technology were cocultured with the 56 kD protein straps obtained from the hippocampus on the 14th day after fimbria transection, the normal hippocampus, and control empty strap respectively, An inverted microscope was used to observe the migration and differentiation of hNSCs. MAP-2 immunofluorescence and AChE histochemistry were used respectively to detect the differentiation of the hNSCs into neurons and AChE-positive neurons on the 21st day after coculture. Then image processing and statistical analysis about the cell number, the cell bodies’areas and the cells’perimeters were carried out. The results showed that the differentiated MAP-2-positive neurons were the best in transected group, the second in normal group, and the worst in control empty group, and all groups showed negative results by AChE histochemistry. The results suggest that differential 56 kD protein obtained from the fimbria-transected hippocampus has an obvious effect on inducing differentiation of hNSCs into MAP-2-positive neurons rather than AChE-positive neurons.