【摘要】 目的:观察原代培养神经细胞对淀粉样蛋白(β-amyloid protein,Aβ)内化,以及星形胶质细胞对这一过程的影响。方法:纯小鼠皮质神经细胞培养14d,分成对照组和Aβ组,各组再分为3个亚组,分别用3种不同浓度的荧光素或荧光素标记的淀粉样蛋白1-42(Aβ1-42-fluo)与神经细胞共孵育24h,在激光扫描共聚焦显微镜下直接观察或进行免疫荧光多重染色后镜下观察,结合图像分析方法分析神经细胞内化Aβ情况及亚细胞结构定位;另取小鼠皮质神经细胞与星形胶质细胞共培养14d,分成对照组和Aβ组,各组分别与200nmol/L荧光素或200nmol/L Aβ1-42-fluo共孵育24h,采用上述方法分析比较星形胶质细胞对神经细胞内化Aβ的影响。结果:荧光素各组细胞内均未见荧光颗粒。培养的纯神经细胞24h内能将浓度为100和200nmol/L Aβ1-42-fluo内化入细胞内,荧光颗粒在神经细胞的胞体和突起均有分布。内化作用与Aβ浓度相关,经免疫荧光方法证明部分内化Aβ位于溶酶体内;在神经细胞与星形胶质细胞共培养组中,神经细胞内化Aβ较纯神经细胞培养组明显增加(P<0.05)。结论:神经细胞能内化一定浓度的Aβ,星形胶质细胞具有促进神经细胞内化Aβ的作用。更多还原

【Abstract】 Objective To observe the internalization of β-amyloid protein(Aβ) in primary cultured neurons and effect of astrocyte on it.Methods The purified cortical neurons of mouse were cultured for 14 d,and were divided into a control group and an Aβ group. Each group was further divided into 3 subgroups. The neurons and 3 different concentration fluorescein or Aβ1-42-fluo were co-incubated for 24 h. The internalization of Aβ and the location of Aβ in subcellular structure were examined by the laser scanning confocal microscope combined with the image analysis method directly or after immunofluorescence staining. Neurons and astrocytes were co-cultured for 14 d. The cultured neurons and astrocytes were divided into a control group and a Aβ group. The cultures were treated with 200 nmol/L fluorescein or 200 nmol/L Aβ1-42-fluo for 24 h respectively. The effect of astrocyte on the internalization was analyzed by the above method. Results There were no fluorescent granules within neurons in every fluorescein group. The purified cortical neurons could internalize 100 nmol/L,or 200 nmol/L Aβ1-42-fluo in 24 h. The fluorescent granules of Aβ1-42 distributed within perikaryon and processes. The internalization was related to the concentration of Aβ. The part of Aβ was located in the lysosome of neurons indicated by immunofluorescence staining.Compared with the purified neurons,the neurons co-cultured with the astrocytes internalized Aβ increased in the internalization of Aβ. There was significant difference between the purified neurons and the co-cultured neurons with astrocytes (P<0.05). Conclusion Neurons could internalize the proper concentration of Aβ. Astrocyte might facilitate the internalization of Aβ in neurons.更多还原