大鼠海马神经干细胞体外增殖、凋亡和分化的激光扫描共焦显微镜观察

目的 采用激光扫描共焦显微镜观察体外培养胎鼠海马神经干细胞（NSCs）增殖、凋亡及其分化情况。方法 体外分离培养胚胎大鼠海马NSCs，把神经干细胞球培养在共焦显微镜专用培养皿中。用 Hoechst 33258染细胞核，免疫荧光细胞化学技术检测巢蛋白（Nestin）的表达以鉴定NSCs，检测神经元、星形胶质细胞的特异性标记物β-Ⅲ型微管蛋白（β-Ⅲ tubulin）和胶质纤维酸性蛋白（GFAP）的表达以测定NSCs的分化能力。通过激光扫描共焦显微镜对神经干细胞球进行光学连续断层扫描，然后用软件进行三维重建，立体动态观察神经球。结果 通过激光扫描共焦显微镜观察到神经球

Observation of proliferation, apoptosis and differentiation of neural stem cells from the hippocampus of fetal rats by laser scanning confocal microscopy

Objective To observe proliferation, apoptosis, and differentiation of cultured neural stem cells（NSCs）from the hippocampus of fetal rats by laser scanning confocal microscopy. Methods NSCs from the hippocampus of fetal rats were isolated and cultured, and the resultant neurospheres grown in petri dishes prior to preparation for confocal microscopy. The nuclei were stained with Hoechst 33258, and an anti-nestin antibody was employed to identify NSCs. Neuron-and astrocyte-specific markers β-III tubulin and glial fibrillary acidic protein（GFAP）were detected to assess NSCs differentiation. Optical continuous tomography of the neurospheres was performed using a laser scanning confocal microscope, and a software was employed to perform three-dimensional reconstruction in order to dynamically observe the neurospheres. Results Nestin-positive neurospheres were observed under a laser scanning confocal microscope. The NSCs gradually developed neurites that intertwined with each other. At the end of 7 days in vitro and twice-passaged, the apoptosis rate was (8.60±2.20)%. After serum-induced differentiation, we observed radial migration of NSCs neurites, which eventually intertwined into a mesh. At the end of 7 days when NSCs from twice-passaged culture were induced to differentiate，the proportions of NSCs that had differentiated into astrocytes and neurons were (68.60±7.64)% and (29.90±8.22)%, respectively（EM>P/EM><0.01）. Conclusion Laser scanning confocal microscopy allows the simultaneous observation of rat neurospheres proliferation, apoptosis, and differentiation, which provides a simple and feasible experimental method for basic NS