【摘要】：目的通過孕酮對(duì)兔脊髓缺血再灌注損傷模型的的干預(yù),研究孕酮對(duì)兔脊髓缺血再灌注損傷的作用。方法采用腹主動(dòng)脈阻斷法誘導(dǎo)兔子的脊髓缺血再灌注模型,其中空白對(duì)照組3只僅進(jìn)行手術(shù)不進(jìn)行主動(dòng)脈的阻斷,隨時(shí)間延長(zhǎng)無明顯病理變化;損傷組15只,手術(shù)后不給于藥物干預(yù);孕酮組15只,分別在術(shù)后0h、24h、48h腹腔注射8mg/kg孕酮。損傷組、孕酮組分別在術(shù)后12h、24h、36h、48h、72h各處死3只兔子。選取術(shù)后24h、48h及72h三個(gè)觀察時(shí)間點(diǎn),采用Tarlov評(píng)分對(duì)兔的后肢神經(jīng)學(xué)功能進(jìn)行評(píng)估。收集兔的脊髓組織樣本,采用HE染色觀察脊髓組織的形態(tài)變化,并對(duì)其神經(jīng)元的保留水平進(jìn)行統(tǒng)計(jì)。采用免疫組化的方法對(duì)脊髓組織中Caspase-8及p53蛋白的表達(dá)水平進(jìn)行檢測(cè),并統(tǒng)計(jì)其中陽(yáng)性細(xì)胞的數(shù)目。收集新鮮的組織樣本采用western blotting技術(shù)對(duì)不同時(shí)間點(diǎn)的p53的蛋白表達(dá)情況進(jìn)行檢測(cè),然后對(duì)其進(jìn)行定量分析。結(jié)果1 HE染色顯示,與空白對(duì)照組相比,損傷組12h無明顯病變,24h脊髓出現(xiàn)出現(xiàn)空泡變性,神經(jīng)元開始皺縮;48h神經(jīng)元大量凋亡,組織出血水腫嚴(yán)重;72h出現(xiàn)炎性細(xì)胞浸潤(rùn);孕酮組在術(shù)后24h到72h內(nèi)也出現(xiàn)損傷組類似的變化,但神經(jīng)元保存數(shù)量較多,且出血水腫及炎性細(xì)胞浸潤(rùn)等病理變化較輕。2術(shù)后12h時(shí),空白對(duì)照組的神經(jīng)元數(shù)量最多,孕酮組次之,損傷組最少;術(shù)后24h時(shí),三組兔子的神經(jīng)元數(shù)量無明顯統(tǒng)計(jì)學(xué)差異(P0.05);術(shù)后48h時(shí),損傷組及孕酮組的神經(jīng)元數(shù)量與空白對(duì)照組相比明顯減少(P0.05),且損傷組的神經(jīng)元數(shù)量明顯的少于孕酮組(P0.05);術(shù)后72h時(shí),損傷組及孕酮組的神經(jīng)元數(shù)量較空白對(duì)照組及術(shù)后24h和48h進(jìn)一步減少(P0.05),且損傷組的神經(jīng)元數(shù)量明顯的小于孕酮組(P0.05)。3術(shù)后12h,損傷組及孕酮組兔子的神經(jīng)功能均發(fā)生不同程度神經(jīng)功能障礙,且損傷組的損傷程度更為嚴(yán)重(P0.05);手術(shù)48h后損傷組的運(yùn)動(dòng)功能較之前顯著降低,孕酮組兔子較術(shù)后24h時(shí)有細(xì)微降低,但仍其功能顯著高于損傷組(P0.05);手術(shù)72h時(shí),損傷組及孕酮組兔子的運(yùn)動(dòng)神經(jīng)功能均有不同程度的降低,但降低程度并無統(tǒng)計(jì)學(xué)差異,且孕酮組的評(píng)分顯著高于損傷組(P0.05)。4三組兔子的脊髓組織的Caspase-8表達(dá)水平進(jìn)行比較發(fā)現(xiàn),12h時(shí),空白對(duì)照組的Caspase-8的陽(yáng)性得分最低,損傷組顯著高于空白對(duì)照組,孕酮組的得分介于兩組之間;48h時(shí),損傷組及孕酮組的Caspase-8的蛋白表達(dá)水平進(jìn)一步提高,且損傷組的表達(dá)水平顯著高于孕酮組;72h時(shí),損傷組的Caspase-8的表達(dá)水平較48h時(shí)進(jìn)一步顯著提高,孕酮組雖有提高,但提高幅度明顯小于損傷組。5對(duì)p53的陽(yáng)性細(xì)胞進(jìn)行統(tǒng)計(jì)發(fā)現(xiàn),24h時(shí),空白對(duì)照組的p53陽(yáng)性得分最低,損傷組為最高,孕酮組的得分介于兩組之間;48h時(shí),損傷組及孕酮組的p53的蛋白表達(dá)水平進(jìn)一步提高,且損傷組的表達(dá)水平顯著高于孕酮組;72h時(shí),損傷組的p53的表達(dá)水平較48h時(shí)進(jìn)一步顯著提高,孕酮組雖有提高,但提高幅度明顯小于損傷組。結(jié)論本研究中,通過對(duì)不同處理狀態(tài)下脊髓缺血再灌注損傷兔子脊髓形態(tài)及炎癥的進(jìn)行評(píng)估,并對(duì)可能的機(jī)制進(jìn)行初步的探索發(fā)現(xiàn):1脊髓缺血再灌注損傷后注射孕酮能夠顯著的減少脊髓組織的炎癥,降低脊髓神經(jīng)元的凋亡;2脊髓缺血再灌注損傷后接受孕酮干預(yù)能夠顯著的提高兔子的運(yùn)動(dòng)神經(jīng)功能的恢復(fù);3脊髓缺血再灌注損傷后接受孕酮干預(yù)能夠顯著降低脊髓組織中Caspase-8及p53蛋白的表達(dá),提示孕酮可能通過降低Caspase-8及p53蛋白的表達(dá)減少脊髓缺血損傷后神經(jīng)元的凋亡。
[Abstract]:Objective To study the effect of progesterone on ischemia-reperfusion injury of spinal cord in rabbits by intervention of progesterone on rabbit spinal cord ischemia-reperfusion injury model. Methods The rabbit spinal cord ischemia/ reperfusion model was induced by abdominal aorta occlusion method, in which 3 groups were only operated without aortic occlusion, no obvious pathological changes were observed over time, 15 were injured in the injury group, no drug intervention was given after the operation, and 15 in the progesterone group. 8mg/ kg progesterone was injected intraperitoneally at 0h, 24h and 48h after operation respectively. Three rabbits were sacrificed at 12h, 24h, 36h, 48h and 72h after operation. The neurological function of the hindlimb of rabbits was evaluated using the lolov score at the three observation time points of 24h, 48h and 72h after operation. The spinal cord tissue samples of rabbits were collected, the morphological changes of spinal cord tissues were observed by HE staining and the retention levels of neurons were counted. The expression level of Caspase-8 and p53 protein in spinal cord tissue was detected by immunohistochemistry and the number of positive cells was counted. The expression of p53 protein in different time points was detected by western blotting technique and quantitative analysis was carried out. Results 1 HE staining showed that there were no obvious pathological changes in 12h of injury group compared with blank control group, vacuous degeneration occurred in 24h spinal cord, and neurons began to collapse; 48h neurons were apoptosis, and the edema of tissue hemorrhage was severe; 72h experienced inflammatory cell infiltration; There were similar changes in the group of progesterone in 24h to 721h after operation, but the number of neurons was much higher, and the pathological changes of hemorrhage edema and inflammatory cell infiltration were lighter. At 12h after operation, the number of neurons in the blank control group was the most, the progesterone group was the second, the injury group was the least, and when the operation was 24h, There was no significant difference in the number of neurons in the three groups (P0.05). When 48h after operation, the number of neurons in the injury group and progesterone group decreased significantly compared with the blank control group (P0.05), and the number of neurons in the injured group was significantly lower than that of the progesterone group (P0.05). The number of neurons in the injury group and progesterone group decreased further (P0.05), and the number of neurons in the injured group was significantly lower than that of the progesterone group (P0.05). The damage level of the injured group was more serious (P0.05), the exercise function of the injured group decreased significantly before the 48h operation, but the level of the progesterone group was significantly lower than that of the injured group at 24h, but its function was significantly higher than that of the injury group (P0.05). The level of Caspase-8 expression in the spinal cord tissue of the three groups of rabbits was significantly higher than that in the injured group (P0.05). The expression level of Caspase-8 in the injury group and progesterone group was higher than that in the control group, and the expression level of Caspase-8 in the injured group was significantly higher than that of the progesterone group at 48h, and the expression level of the injured group was significantly higher than that of the progesterone group. The expression level of Caspase-8 in the injured group was further improved when the expression level of Caspase-8 was higher than that in the injured group, but the increase in the progesterone group was significantly less than that of the injury group. The positive cells of p53 were statistically found to be the lowest in the blank control group and the highest in the injury group at 24h. The expression level of p53 in the injury group and the progesterone group was further increased after 48h, and the expression level of the injured group was significantly higher than that of the progesterone group at 48h. The expression level of p53 in the injured group was further improved when the expression level of p53 in the injured group was higher than that of the progesterone group at 48h. but the increase in amplitude is significantly less than that of the injury group. Conclusion In this study, the spinal cord morphology and inflammation of rabbits were assessed by ischemia-reperfusion injury of spinal cord in different treatment states, and the possible mechanisms were preliminarily explored. reducing the apoptosis of spinal cord neurons; receiving progesterone intervention after spinal ischemic reperfusion injury to remarkably improve the recovery of motor nerve function of rabbits; 3, receiving progesterone intervention after spinal ischemia reperfusion injury to remarkably reduce the expression of Caspase-8 and p53 protein in spinal cord tissues, It is suggested that progesterone may decrease the apoptosis of neurons after spinal ischemic injury by reducing the expression of Caspase-8 and p53 protein.
【學(xué)位授予單位】：華北理工大學(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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