大鼠損傷脊髓SKIP表達(dá)的時(shí)空變化規(guī)律及作用
發(fā)布時(shí)間：2018-08-24 11:53
【摘要】：目的脊髓損傷后機(jī)體病變及修復(fù)過(guò)程中常伴隨著一系列病理生理變化,這些病理生理變化與一些細(xì)胞、分子的變化息息相關(guān)。本研究通過(guò)建立動(dòng)物模型及實(shí)驗(yàn)觀察探究SKIP(Ski interacting protein,ski相關(guān)蛋白)在大鼠正常及損傷后的脊髓中表達(dá)的時(shí)空變化規(guī)律,及其SKIP在脊髓損傷及其康復(fù)過(guò)程中的作用。方法將90只成年雌性Sprague-Dawley大鼠按照隨機(jī)數(shù)字表法隨機(jī)分成兩組,即假手術(shù)組(n=45)和打擊組(n=45),每組內(nèi)部各設(shè)五個(gè)時(shí)間點(diǎn)(1天、3天、5天、7天、14天),每個(gè)時(shí)間點(diǎn)各9只大鼠。建立動(dòng)物模型時(shí)假手術(shù)組只咬開(kāi)椎板,不予打擊脊髓;打擊組咬開(kāi)椎板顯露脊髓,并用Allen法制作T10打擊損傷模型,打擊強(qiáng)度定為10g×25 mm。術(shù)后兩組大鼠在每個(gè)時(shí)間點(diǎn)均各進(jìn)行一次BBB后肢功能評(píng)分,用以評(píng)估大鼠的行為功能。評(píng)分完成后每一時(shí)間點(diǎn)分別選取3只大鼠,取其脊髓組織做成冰凍切片后進(jìn)行尼氏染色,觀察損傷后脊髓神經(jīng)元的病理變化。每組每個(gè)時(shí)間點(diǎn)各另取3只大鼠的脊髓組織切片后行免疫熒光染色,以觀察正常及損傷脊髓組織中SKIP的表達(dá)情況。每組每個(gè)時(shí)間點(diǎn)剩余的3只大鼠提取脊髓組織蛋白后進(jìn)行Western Blot檢測(cè)以明確脊髓組織中目標(biāo)蛋白(SKIP)的表達(dá)情況。結(jié)果1、BBB評(píng)分結(jié)果顯示:打擊組各時(shí)間點(diǎn)BBB評(píng)分均低于假手術(shù)組(n=9,P0.01)。2、尼氏染色結(jié)果表明:與假手術(shù)組相比,打擊后5d脊髓神經(jīng)元胞漿中尼氏小體開(kāi)始崩解、凝聚、分布不規(guī)則,神經(jīng)元變性壞死,14d時(shí)尼氏小體大部分崩解、凝聚,損傷進(jìn)一步加重。3、熒光免疫組化染色顯示,SKIP主要在脊髓灰質(zhì)中表達(dá),白質(zhì)中極少表達(dá);損傷后SKIP表達(dá)呈現(xiàn)先上升后下降的趨勢(shì),5d時(shí)達(dá)到高峰,14d時(shí)明顯降低;光密度分析顯示,與假手術(shù)組比較,打擊組SKIP表達(dá)明顯增高(n=3,P0.05);雙標(biāo)結(jié)果顯示,損傷后脊髓灰質(zhì)中,SKIP與神經(jīng)元特異性標(biāo)記物Neu N顯示出共表達(dá)信號(hào),而與膠質(zhì)纖維的特異性標(biāo)記物GFAP無(wú)共表達(dá)信號(hào)。4、Western Blot結(jié)果顯示,與假手術(shù)組比較,脊髓損傷后1d、3d、5d、7d、14d目標(biāo)蛋白(SKIP)的表達(dá)呈現(xiàn)先上升后下降的趨勢(shì),與免疫熒光的結(jié)果基本一致。結(jié)論本研究初步探索了正常及損傷脊髓組織中SKIP表達(dá)的位置、以及脊髓損傷后SKIP的表達(dá)隨著時(shí)間推移而呈現(xiàn)出的變化規(guī)律,結(jié)合脊髓神經(jīng)元及其脊髓神經(jīng)元中尼氏小體的病理變化,我們推斷,SKIP可能是一種作用于神經(jīng)元并影響其凋亡的新的信號(hào)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2
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