原子力显微镜下观察急性压力损伤后脊髓背根神经节神经元的结构变化

Structural changes of dorsal root ganglion neuron injured by acute mechanical pressure: an AFM observation

中文关键词:  脊髓损伤  原子力显微镜  神经元细胞  细胞培养  机械压力

英文关键词:Spinal cord injury  Mechanical pressure  Cell culture  Neuron cells  Atomic force microscope

中文摘要:

  【摘要】 目的：利用脊髓背根神经节神经元细胞体外机械压力损伤模型，探计单纯机械压力因素对背根神经节神经元细胞的损伤效应，尤其是细胞膜骨架蛋白的变化情况。方法：新生SD大鼠脊髓背根神经节神经元体外原代培养，利用多功能全自动细胞压力仪（国家实用新型专利ZL 200820030390.7），将培养细胞分为A、B、C、D四组，其中A组为未施加机械压力损伤的对照组，B、C、D组分别给予0.3mPa、0.5mPa.、0.7mPa的压力损伤10min，每组观察细胞爬片6个，于加压后培养8h取细胞爬片固定，送原子力显微镜检测，观察神经元细胞的细胞膜骨架蛋白纳米级变化情况，获取并分析胞膜表面颗粒高度分布数据及原子力显微镜图片。结果：对照组细胞膜表面平滑，无明显粗糙颗粒；0.3mPa组细胞膜表面颗粒突起较对照组增多，膜颗粒总体高度增加，局部变粗糙，胞膜上蛋白高度分布直方图峰值右移；0.5mPa组较0.3mPa组胞膜粗糙度更加明显；0.7mPa组膜表面蛋白颗粒总体高度较0.3mPa及0.5mPa组降低，粗糙颗粒减少，但出现胞膜骨架蛋白解聚与构像变化，胞膜出现孔道样改变。胞膜颗粒高度分布峰值、分布宽度及最高颗粒值的单因素方差分析显示，压力损伤各组间及损伤组与对照组均有统计学差异（P＜0.01）。结论：机械压力损伤因素可引起细胞膜骨架蛋白变化和表面颗粒高度变化，为压力损伤的细胞保护研究提供了实验数据。

英文摘要:

  【Abstract】 Objectives: To investigate the spinal cord injury mechanism especially the nanoscale reaction of membrane skeleton protein in spinal cord injury under mechanical pressure by using the cultured dorsal root ganglion neuron cells in vitro and simulation of mechanical injury device. Methods: The primary culturing of dorsal root ganglion neuron cells from new born S-D rats wsa used, and the mechanical pressure on cultured cells wsa added by the multi-functional automatically mechanical pressure equipment(national patent of utility model, ZL 200820030390.7). The cells were divided to 4 groups, group A, B, C and D. The group A was tested as control group. The group B, C and D experienced 0.3mPa, 0.5mPa and 0.7mPa mechanical pressure for 10 min respectively. The cells were fixed by formaldehyde when cultured for 8 hours, the ultra structure of the cell membrane skeleton protein was observed by AFM(atom force microscope), with 6 different Petri dishes of cells for each group. The nanoscale reaction of membrane skeleton protein wsa observed, the corresponding data of the height of the membrane particle and roughness parameters as well as the AFM photos were acquired. Results: The cell membrane of the control group was smooth and not apparently rough. The 0.3mPa group showed the particle height of cell membrane increased apparently compared with the control group, the membrane surface seemed more rough, and the peak value of the histogram of the protein height on cell surface shifted rightly. The 0.5mPa group showed more severe changes than the 0.3mPa group. The 0.7mpa group showed the particle height of cell membrane protein decreased and became less rough, nevertheless, some porous channel occurred at the membrane. The statistical results of the peak value, the maximal value and the width of particle height value indicated that different mechanical pressure value produced different membrane roughness(P<0.01). Conclusions: Different mechanical pressures produce different membrane roughness as well as the membrane skeleton protein, which can supply basis for protection of injured cells.