Ski在大鼠脊髓损伤后的时空表达规律研究

发布时间：2018-05-23 18:35

本文选题：脊髓损伤 + 行为学　；参考：《兰州大学》2017年硕士论文

【摘要】：第一部分大鼠脊髓损伤后的行为学及病理学变化规律研究目的建立科学可靠的脊髓损伤(Spinal Cord Injury,SCI)模型并明确SCI后大鼠行为学及病理学变化规律,为下一步研究奠定基础。方法30只成年雌性Sprague-Dawley(SD)大鼠,随机分为假手术组(n=15)和打击组(n=15),各组内部设五个时间点(1周、2周、4周、8周、12周),每个时间点3只大鼠。用Allen法制备T10打击损伤模型,打击强度为10 g×25 mm。损伤后1 d、3 d、1周、2周、4周、8周、12周时各行一次行BBB后肢功能评分;术后1周、2周、4周、8周、12周时,各组各时间点分别取大鼠脊髓行HE染色,观察损伤后脊髓病理变化及空洞形成规律。结果1、损伤组各时间点BBB评分均低于假手术组(n=3,P0.05)。2、与假手术组相比,打击后1周、2周损伤脊髓主要以坏死为主;4周时,空洞完全形成,空洞周围有致密瘢痕组织;8周、12周空洞及瘢痕无明显变化,但损伤中心及附近脊髓明显变细。3、通过BBB评分和HE染色结果,对比以往研究结果,充分明确脊髓打击模型建立成功。结论本实验通过行为学和病理学评估,成功建立了科学可靠的SCI模型,并明确了SCI后大鼠的行为学变化和脊髓病理改变的规律,为下一步实验奠定了基础。第二部分Ski在大鼠脊髓损伤后的表达变化规律研究目的初步研究Ski在大鼠SCI后随时间的表达变化规律。方法60只成年雌性SD大鼠,分组同前,每组设1周、2周、4周、8周、12周五个时间点,每个时间点6只大鼠。同前方法制作SCI模型并进行BBB评分,在设定的时间点各组均取6只大鼠,其中3只用于损伤上端及损伤中心的免疫荧光单标检测和荧光半定量分析,另外3只行Western Blot检测,观察Ski的表达变化规律。结果1、BBB评分结果同前。2、免疫荧光及半定量分析显示,ski在正常脊髓中表达较低,损伤后ski表达逐渐增高,8周时达到高峰,12周时有所降低;与假手术组比较,1周组增高无统计学意义(n=3,P0.05);其余各组ski表达明显增高(n=3,P0.001);同时,ski在正常及损伤后12周脊髓中主要分布于白质,灰质中表达较少;损伤后2周、4周和8周时灰质中出现了明确的ski表达信号。3、损伤中心免疫荧光显示,ski在空洞周围密集表达,并形成明显高表达带。4、Western Blot检测显示,相比假手术组,损伤后1周时Ski表达无增高;第2周时,Ski表达开始增高,4周时增高明显增高,8周和12周时表达达到高峰,并呈稳定趋势。结论通过本部分研究,我们首次明确了Ski在SCI后灰质及白质中的分布规律及随时间的表达变化规律,并且发现了Ski在损伤空洞周围密集高表达的现象,提示Ski可能与胶质瘢痕的形成有一定关系。第三部分Ski在大鼠脊髓损伤后表达的细胞定位研究目的进一步明确Ski在SCI后表达的细胞定位规律,并分析Ski在大鼠SCI过程中可能发挥的作用。方法60只成年雌性SD大鼠,分组同前,每组设1周、2周、4周、8周、12周五个时间点,每个时间点6只大鼠。同前方法制作SCI模型并进行BBB评分,在设定的时间点各组均取6只大鼠,其中3只用于免疫荧光双标标染色,另外3只行胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)表达的Western Blot检测,并对Ski表达、GFAP表达及BBB评分做相关性分析。结果1、BBB评分结果同前。2、假手术组和打击组各时间点Ski和Neu N免疫荧光双标染色没有发现二者明确的共表达信号;Ski和Nestin荧光双标同样没有发现二者的共表达信号;但是,Ski和GFAP免疫荧光双标染色发现,在假手术组和打击组各时间点均发现了明确的共表达信号,并且发现这种共表达信号在损伤空洞周围明显增强,并从第4周开始趋于稳定。3、GFAP的Western Blot检测发现相较于假手术组,损伤后1周和2周GFAP表达无明显增高,4周时其表达明显增高,8周、12周时基本趋于稳定。4、Ski、GFAP、BBB评分三者之间的相关性分析发现,Ski表达与GFAP表达、GFAP表达与BBB评分之间均呈明确的正相关关系。结论本部分研究首次明确了SCI后Ski主要表达于星形胶质细胞,而在神经元和神经干细胞中无明确表达;同时Ski的表达与星形胶质细胞的活化增殖及胶质瘢痕的形成有明确的相关性;结合前两部分实验,我们推测Ski可能是调控反应性星形胶质增生及胶质瘢痕形成过程的新的作用分子。
[Abstract]:A study on the behavioral and pathological changes of spinal cord injury in the first part of the rats: Objective To establish a scientific and reliable Spinal Cord Injury (SCI) model and to clarify the behavior and pathological changes of the rats after SCI, and lay the foundation for the next step of the study. Methods 30 adult female Sprague-Dawley (SD) rats were randomly divided into sham operation. Group (n=15) and attack group (n=15), each group had five time points (1 weeks, 2 weeks, 4 weeks, 8 weeks, 12 weeks), 3 rats at each time point. The T10 strike damage model was prepared by Allen method. The attack intensity was 1 D, 3 D, 1 week, 2 weeks, 1 weeks, week, week, week, week, week, week, week, week, week, week, every time. HE staining in the spinal cord of rats at each time point was taken respectively to observe the pathological changes of the spinal cord and the formation of cavities in the spinal cord. Results 1, the BBB score at each time point of the injury group was lower than that of the sham operation group (n=3, P0.05).2. Compared with the sham operation group, the injured spinal cord was mainly caused by the necrosis at 2 weeks after the attack, and at the 4 week, the cavity was completely formed and the cavity around the cavity was dense. There was no obvious change in cicatricial tissue and cicatricial scar in 8 weeks and 12 weeks, but the damage center and nearby spinal cord were obviously thinner.3. Through the results of BBB score and HE staining, the results of the previous study were compared, and the successful model of spinal cord percussion was established. Conclusion this experiment has successfully established a scientific and reliable SCI model through behavioral and pathological evaluation. The change of behavior and pathological changes of spinal cord after SCI in rats laid the foundation for the next experiment. Second part of the expression of Ski after spinal cord injury in rats was used to study the change regularity of the expression of Ski in rats after SCI. Methods 60 adult female SD rats were divided into groups at the same time, each group was set up for 1 weeks, 2 weeks, 4 weeks. 8 weeks, 12 Friday time points, 6 rats at each time point. The SCI model was made with the former method and the BBB score was made. 6 rats were taken at the set time point, of which 3 were used for the immunofluorescence single mark detection and the semi quantitative analysis of the damage center and the damage center, and the other 3 were detected by Western Blot. The expression of Ski was observed. Results 1, the results of BBB score were same as.2, immunofluorescence and semi quantitative analysis showed that the expression of ski in the normal spinal cord was lower, the expression of ski increased gradually after the injury, reached the peak at 8 weeks and decreased at 12 weeks. Compared with the sham operation group, the increase of the group was not statistically significant (n=3, P0.05), and the expression of ski in the other groups was significantly increased (n=3, P0.001); SK (n=3, P0.001); SK, and sk. I was mainly distributed in white matter and gray matter in 12 weeks after normal and injury. 2 weeks after injury, a clear ski expression signal.3 was found in the gray matter at 4 and 8 weeks. The central immunofluorescence showed that ski was densely expressed around the cavity, and the high expression of.4 was formed, and the Western Blot detection showed that compared to the sham operation group, 1 after the injury, 1 At the second week, the expression of Ski increased and increased obviously at 4 weeks, and the expression reached a peak at 8 and 12 weeks at the second week. Conclusion the distribution of Ski in gray matter and white matter after SCI and the variation of the expression with time were clearly defined by this part of the study. And we found that Ski was damaged in the cavity. The dense and high expression of Ski may have a certain relationship with the formation of glial scar. Third the localization of Ski after spinal cord injury in rats was used to further clarify the localization of Ski after SCI, and to analyze the role of Ski in the SCI process in rats. Method 60 adult female SD rats In the same group, each group had 1 weeks, 2 weeks, 4 weeks, 8 weeks, 12 Friday time and 6 rats at each time point. The SCI model was made with the former method and the BBB score was made. 6 rats were taken at the set time points, 3 of them were used for immunofluorescence double labeling, and the other 3 was Glial Fibrillary Acidic Protein, GFAP). The expression of Western Blot was detected and the expression of Ski, GFAP expression and BBB score were analyzed. Results 1, the results of BBB score were the same as the former.2. No clear co expression signal was found in the two cases of Ski and Neu N in the sham operation group and the attack group at each time point; Ski and Nestin fluorescence double standard did not find the co expression signal of the two. However, Ski and GFAP immunofluorescence staining showed that a clear co expression signal was found at all time points in the sham operation group and the attack group, and it was found that the co expression signal was obviously enhanced around the damaged cavity and tended to stabilize.3 from fourth weeks. The Western Blot detection of GFAP was found to be compared to the sham operation group, and 1 weeks and 2 weeks after the injury, G was found. The expression of FAP was not significantly increased, the expression of 4 Zhou Shiqi was significantly higher, 8 weeks, and at 12 weeks, basically tending to stable.4, Ski, GFAP, BBB score between three and the correlation analysis found that the expression of Ski and GFAP, GFAP expression and BBB score were clearly positive correlation. Conclusion this part of the study was first clear that SCI Ski was mainly expressed in astrocytes. There is no clear expression in neurons and neural stem cells, and there is a clear correlation between the expression of Ski and the activation and proliferation of astrocytes and the formation of glial scar. Combined with the previous two experiments, we speculate that Ski may be a new molecule that regulates the process of reactive astrogliosis and gelatin scar formation.
【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

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