大鼠损伤脊髓SKIP表达的时空变化规律及作用

发布时间：2018-08-24 11:53

【摘要】：目的脊髓损伤后机体病变及修复过程中常伴随着一系列病理生理变化,这些病理生理变化与一些细胞、分子的变化息息相关。本研究通过建立动物模型及实验观察探究SKIP(Ski interacting protein,ski相关蛋白)在大鼠正常及损伤后的脊髓中表达的时空变化规律,及其SKIP在脊髓损伤及其康复过程中的作用。方法将90只成年雌性Sprague-Dawley大鼠按照随机数字表法随机分成两组,即假手术组(n=45)和打击组(n=45),每组内部各设五个时间点(1天、3天、5天、7天、14天),每个时间点各9只大鼠。建立动物模型时假手术组只咬开椎板,不予打击脊髓;打击组咬开椎板显露脊髓,并用Allen法制作T10打击损伤模型,打击强度定为10g×25 mm。术后两组大鼠在每个时间点均各进行一次BBB后肢功能评分,用以评估大鼠的行为功能。评分完成后每一时间点分别选取3只大鼠,取其脊髓组织做成冰冻切片后进行尼氏染色,观察损伤后脊髓神经元的病理变化。每组每个时间点各另取3只大鼠的脊髓组织切片后行免疫荧光染色,以观察正常及损伤脊髓组织中SKIP的表达情况。每组每个时间点剩余的3只大鼠提取脊髓组织蛋白后进行Western Blot检测以明确脊髓组织中目标蛋白(SKIP)的表达情况。结果1、BBB评分结果显示:打击组各时间点BBB评分均低于假手术组(n=9,P0.01)。2、尼氏染色结果表明:与假手术组相比,打击后5d脊髓神经元胞浆中尼氏小体开始崩解、凝聚、分布不规则,神经元变性坏死,14d时尼氏小体大部分崩解、凝聚,损伤进一步加重。3、荧光免疫组化染色显示,SKIP主要在脊髓灰质中表达,白质中极少表达;损伤后SKIP表达呈现先上升后下降的趋势,5d时达到高峰,14d时明显降低;光密度分析显示,与假手术组比较,打击组SKIP表达明显增高(n=3,P0.05);双标结果显示,损伤后脊髓灰质中,SKIP与神经元特异性标记物Neu N显示出共表达信号,而与胶质纤维的特异性标记物GFAP无共表达信号。4、Western Blot结果显示,与假手术组比较,脊髓损伤后1d、3d、5d、7d、14d目标蛋白(SKIP)的表达呈现先上升后下降的趋势,与免疫荧光的结果基本一致。结论本研究初步探索了正常及损伤脊髓组织中SKIP表达的位置、以及脊髓损伤后SKIP的表达随着时间推移而呈现出的变化规律,结合脊髓神经元及其脊髓神经元中尼氏小体的病理变化,我们推断,SKIP可能是一种作用于神经元并影响其凋亡的新的信号分子。
[Abstract]:Objective following spinal cord injury, a series of pathological and physiological changes are often accompanied by pathological changes, which are closely related to the changes of some cells and molecules. The purpose of this study was to investigate the temporal and spatial changes of SKIP (Ski interacting protein,ski related protein expression in normal and injured spinal cord of rats and the role of SKIP in spinal cord injury and rehabilitation. Methods 90 adult female Sprague-Dawley rats were randomly divided into two groups according to random number table: sham operation group (nong45) and attack group (nong45). Each group was divided into five time points (1 day, 3 days, 5 days, 7 days, 14 days), each time point was 9 rats. When the animal model was established, the sham operation group only opened the lamina and did not attack the spinal cord, while the attack group opened the lamina and exposed the spinal cord, and made the T10 injury model with the Allen method. The strike intensity was 10 g 脳 25 mm.. The functional scores of BBB hind limbs were used to evaluate the behavioral function of the rats in each time point after operation. Three rats were selected at each time point after the score was finished. The spinal cord tissue was made into frozen sections and stained with Nissl staining to observe the pathological changes of spinal cord neurons after injury. The expression of SKIP in normal and injured spinal cord tissues was observed by immunofluorescence staining after the spinal cord sections of 3 other rats were taken from each group at each time point. The remaining 3 rats in each group were extracted from spinal cord tissue protein and detected by Western Blot to determine the expression of target protein (SKIP) in spinal cord tissue. Results 1 the results of BBB score showed that the BBB score of the attack group was lower than that of the sham operation group at all time points (NN9 / P0.01). The results of Nissl staining showed that the Nissl corpuscles in the spinal cord neurons began to disintegrate, coagulate and distribute irregularly 5 days after the attack compared with the sham-operated group. After 14 days of degeneration, necrosis, neuronal degeneration and necrosis, most of the Nissl corpuscles were disintegrated, condensed, and the damage was further aggravated. Fluorescence immunohistochemical staining showed that the expression of sips was mainly in the gray matter of the spinal cord, but rarely in the white matter. After injury, the expression of SKIP increased first and then decreased. Optical density analysis showed that the expression of SKIP was significantly higher in the attack group than that in the sham-operated group (P 0.05), and the double labeling showed that the expression of SKIP in the attack group was significantly higher than that in the sham operation group (P 0.05). After injury, spp showed coexpression signal with neuron specific marker Neu N, but no co-expression signal with glial fiber specific marker GFAP. The results showed that compared with sham operation group, Skip showed no co-expression signal. After spinal cord injury, the expression of target protein (SKIP) increased first and then decreased, which was consistent with the result of immunofluorescence. Conclusion this study preliminarily explored the position of SKIP expression in normal and injured spinal cord tissues, and the changes of SKIP expression over time after spinal cord injury. According to the pathological changes of spinal neurons and their bodies, we infer that Skip may be a new signal molecule acting on neurons and affecting their apoptosis.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

【相似文献】