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

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

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Hoechst33258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-Ⅲtubulin)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Nestin阳性表达,NSCs的突起随着发育逐渐增长,并相互缠绕,经2次传代后培养7d的NSCs凋亡率为(8.60±2.20)%。在血清诱导下分化后的细胞突起从细胞球向四周呈放射状迁移,且突起相互交错成网状。经2次传代后诱导分化7 d的NSCs分化为星形胶质细胞和神经元的比例分别为(68.60±7.64)%和(29.90±8.22)%(P<0.01)。结论对大鼠神经干细胞球的增殖、凋亡及分化的观察激光扫描共焦显微镜发挥了独特的优势。

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 p-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 eonfoeal 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 astroeytes and neurons were （68.60 ± 7.64）% and （29.90 ± 8.22） % , respectively （ P 〈 0.01 ）. Conclusion Laser scanning confoeal microscopy allows the simultaneous observation of rat neurospheres proliferation, apoptosis, and differentiation, which provides a simple and feasible experimental method for basic NSCs research.