本文選題：脊髓損傷 + 行為學(xué)��； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：第一部分大鼠脊髓損傷后的行為學(xué)及病理學(xué)變化規(guī)律研究目的建立科學(xué)可靠的脊髓損傷(Spinal Cord Injury,SCI)模型并明確SCI后大鼠行為學(xué)及病理學(xué)變化規(guī)律,為下一步研究奠定基礎(chǔ)。方法30只成年雌性Sprague-Dawley(SD)大鼠,隨機分為假手術(shù)組(n=15)和打擊組(n=15),各組內(nèi)部設(shè)五個時間點(1周、2周、4周、8周、12周),每個時間點3只大鼠。用Allen法制備T10打擊損傷模型,打擊強度為10 g×25 mm。損傷后1 d、3 d、1周、2周、4周、8周、12周時各行一次行BBB后肢功能評分;術(shù)后1周、2周、4周、8周、12周時,各組各時間點分別取大鼠脊髓行HE染色,觀察損傷后脊髓病理變化及空洞形成規(guī)律。結(jié)果1、損傷組各時間點BBB評分均低于假手術(shù)組(n=3,P0.05)。2、與假手術(shù)組相比,打擊后1周、2周損傷脊髓主要以壞死為主;4周時,空洞完全形成,空洞周圍有致密瘢痕組織;8周、12周空洞及瘢痕無明顯變化,但損傷中心及附近脊髓明顯變細(xì)。3、通過BBB評分和HE染色結(jié)果,對比以往研究結(jié)果,充分明確脊髓打擊模型建立成功。結(jié)論本實驗通過行為學(xué)和病理學(xué)評估,成功建立了科學(xué)可靠的SCI模型,并明確了SCI后大鼠的行為學(xué)變化和脊髓病理改變的規(guī)律,為下一步實驗奠定了基礎(chǔ)。第二部分Ski在大鼠脊髓損傷后的表達(dá)變化規(guī)律研究目的初步研究Ski在大鼠SCI后隨時間的表達(dá)變化規(guī)律。方法60只成年雌性SD大鼠,分組同前,每組設(shè)1周、2周、4周、8周、12周五個時間點,每個時間點6只大鼠。同前方法制作SCI模型并進(jìn)行BBB評分,在設(shè)定的時間點各組均取6只大鼠,其中3只用于損傷上端及損傷中心的免疫熒光單標(biāo)檢測和熒光半定量分析,另外3只行Western Blot檢測,觀察Ski的表達(dá)變化規(guī)律。結(jié)果1、BBB評分結(jié)果同前。2、免疫熒光及半定量分析顯示,ski在正常脊髓中表達(dá)較低,損傷后ski表達(dá)逐漸增高,8周時達(dá)到高峰,12周時有所降低;與假手術(shù)組比較,1周組增高無統(tǒng)計學(xué)意義(n=3,P0.05);其余各組ski表達(dá)明顯增高(n=3,P0.001);同時,ski在正常及損傷后12周脊髓中主要分布于白質(zhì),灰質(zhì)中表達(dá)較少;損傷后2周、4周和8周時灰質(zhì)中出現(xiàn)了明確的ski表達(dá)信號。3、損傷中心免疫熒光顯示,ski在空洞周圍密集表達(dá),并形成明顯高表達(dá)帶。4、Western Blot檢測顯示,相比假手術(shù)組,損傷后1周時Ski表達(dá)無增高;第2周時,Ski表達(dá)開始增高,4周時增高明顯增高,8周和12周時表達(dá)達(dá)到高峰,并呈穩(wěn)定趨勢。結(jié)論通過本部分研究,我們首次明確了Ski在SCI后灰質(zhì)及白質(zhì)中的分布規(guī)律及隨時間的表達(dá)變化規(guī)律,并且發(fā)現(xiàn)了Ski在損傷空洞周圍密集高表達(dá)的現(xiàn)象,提示Ski可能與膠質(zhì)瘢痕的形成有一定關(guān)系。第三部分Ski在大鼠脊髓損傷后表達(dá)的細(xì)胞定位研究目的進(jìn)一步明確Ski在SCI后表達(dá)的細(xì)胞定位規(guī)律,并分析Ski在大鼠SCI過程中可能發(fā)揮的作用。方法60只成年雌性SD大鼠,分組同前,每組設(shè)1周、2周、4周、8周、12周五個時間點,每個時間點6只大鼠。同前方法制作SCI模型并進(jìn)行BBB評分,在設(shè)定的時間點各組均取6只大鼠,其中3只用于免疫熒光雙標(biāo)標(biāo)染色,另外3只行膠質(zhì)纖維酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)表達(dá)的Western Blot檢測,并對Ski表達(dá)、GFAP表達(dá)及BBB評分做相關(guān)性分析。結(jié)果1、BBB評分結(jié)果同前。2、假手術(shù)組和打擊組各時間點Ski和Neu N免疫熒光雙標(biāo)染色沒有發(fā)現(xiàn)二者明確的共表達(dá)信號;Ski和Nestin熒光雙標(biāo)同樣沒有發(fā)現(xiàn)二者的共表達(dá)信號;但是,Ski和GFAP免疫熒光雙標(biāo)染色發(fā)現(xiàn),在假手術(shù)組和打擊組各時間點均發(fā)現(xiàn)了明確的共表達(dá)信號,并且發(fā)現(xiàn)這種共表達(dá)信號在損傷空洞周圍明顯增強,并從第4周開始趨于穩(wěn)定。3、GFAP的Western Blot檢測發(fā)現(xiàn)相較于假手術(shù)組,損傷后1周和2周GFAP表達(dá)無明顯增高,4周時其表達(dá)明顯增高,8周、12周時基本趨于穩(wěn)定。4、Ski、GFAP、BBB評分三者之間的相關(guān)性分析發(fā)現(xiàn),Ski表達(dá)與GFAP表達(dá)、GFAP表達(dá)與BBB評分之間均呈明確的正相關(guān)關(guān)系。結(jié)論本部分研究首次明確了SCI后Ski主要表達(dá)于星形膠質(zhì)細(xì)胞,而在神經(jīng)元和神經(jīng)干細(xì)胞中無明確表達(dá);同時Ski的表達(dá)與星形膠質(zhì)細(xì)胞的活化增殖及膠質(zhì)瘢痕的形成有明確的相關(guān)性;結(jié)合前兩部分實驗,我們推測Ski可能是調(diào)控反應(yīng)性星形膠質(zhì)增生及膠質(zhì)瘢痕形成過程的新的作用分子。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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