本文選題：IL-17 + STAT3��； 參考：《廣西醫(yī)科大學》2015年碩士論文

【摘要】：脊髓損傷(Spinal cord injury, SCI)是指脊髓受到外力的撞擊后導致的完全性或不完全性脊髓損傷平面以下的運動、感覺等功能障礙,是脊柱損傷中最嚴重的并發(fā)癥。最新的統(tǒng)計數(shù)字表明,美國每年約有25萬人患不同程度的SCI,年發(fā)生率為28-50/100萬人,我國上海SCI的年發(fā)生率約為13.7/100萬人以上。而且隨著世界各國經(jīng)濟水平的發(fā)展,脊髓損傷發(fā)生率也呈現(xiàn)逐年增高的趨勢。針對脊髓損傷后導致的功能障礙目前國內(nèi)外尚無有明確療效的治療手段,因此研究有效的治療SCI的方法將有巨大的經(jīng)濟和社會效益。目前研究表明Th17細胞在自身免疫介導的組織損傷、器官特異自身免疫反應中起著關鍵的作用。在實驗性自身免疫性腦脊髓炎(Experimental autoimmune encephalomyelitis, EAE)小鼠富含產(chǎn)生IL-17的CD4+T細胞轉(zhuǎn)輸給正常小鼠后可誘導出嚴重的EAE,并且疾病的嚴重程度與IL-17的水平相關。另外在研究SCI后T細胞亞群的變化時發(fā)現(xiàn),抗炎細胞Th2/Treg和促炎細胞Th1/Th17均有激活,提示Th17細胞的效應分子IL-17A可能參與了SCI的繼發(fā)性脊髓損傷過程。但在急性脊髓損傷炎癥反應中IL-17細胞因子免疫發(fā)生機制如何?相關細胞因子變化及其相互關系如何?目前尚未完全闡明。為了進一步闡明急性脊髓損傷炎癥反應中IL-17細胞因子免疫發(fā)生機制,為治療急性脊髓損傷尋求新的治療靶點,本課題建立急性脊髓損傷大鼠模型,進行以下2方面研究。第一部分,急性脊髓損傷大鼠模型不同時期IL-17、IL-23表達的研究。75只成年健康雄性SD大鼠隨機分為對照組(15只)和SCI組(60只)。SCI組分成1h、24h、48h及72h四個時間亞組(各15只)。采用改良Allen's重物打擊法成功建立大鼠急性脊髓損傷模型,模型制備前一天及術(shù)后1h、24h、48h及72h運用BBB評分檢測大鼠后肢運動功能。HE染色法檢測觀察脊髓損傷后脊髓的病理改變,免疫組織化學染色法檢測大鼠脊髓組織中IL-17的表達水平。采用反轉(zhuǎn)錄-聚合酶鏈反應(RT-PCR)法檢測各組大鼠脾臟組織中IL-17和IL-23的mRNA表達水平。HE染色示脊髓損傷后脊髓組織形態(tài)結(jié)構(gòu)紊亂,打擊處白質(zhì)、灰質(zhì)有明顯的壓縮跡象,中央管被明顯破壞,毛線血管充血、細胞水腫、炎性細胞浸潤、細胞凋亡等現(xiàn)象在脊髓損傷各個時間亞組呈現(xiàn)出由輕微至嚴重的趨勢。RT-PCR結(jié)果發(fā)現(xiàn)：與對照組大鼠相比,脊髓損傷組大鼠脾組織中IL-17mRNA、IL-23mRNA表達量顯著上升,具有統(tǒng)計學意義。免疫組織化學染色發(fā)現(xiàn)脊髓組織中IL-17在對照組中幾乎不表達,損傷1h后開始稍微表達,24h后上升表達達到峰值,損傷48h、72h后IL-17的表達逐漸下降。第二部分,急性脊髓損傷大鼠STAT3信號途徑介導的IL-17相關細胞因子的分化機制研究。本部分包括兩個方面的研究：1、脊髓損傷后大鼠脊髓和脾臟組織STAT3的表達研究；2、大鼠脊髓損傷后調(diào)控大鼠IL-17分化的相關因子的血清濃度鑒定。在脊髓損傷后大鼠脊髓和脾臟組織STAT3的表達研究中,采用Western blot法檢測SCI大鼠脊髓、脾臟組織中的p-STAT3的相對表達量,與對照組比較脊髓損傷組大鼠脊髓、脾臟組織中p-STAT3的表達量顯著升高以24h組上升最為明顯。大鼠脊髓損傷后調(diào)控大鼠IL-17分化的相關因子的血清濃度鑒定中,采用ELISA法檢測SCI大鼠外周血上清液中的IL-6、IL-21及IL-23的表達水平。結(jié)果發(fā)現(xiàn),與對照組比較脊髓損傷組大鼠血清中IL-6、 IL-21及IL-23的表達量顯著上升以24h組上升最為明顯。本課題研究結(jié)果提示：(1)SCI大鼠隨著急性脊髓損傷時間的延長脊髓組織呈現(xiàn)出出血范圍擴增、炎癥反應加劇、神經(jīng)元死亡數(shù)量增加等病理改變。(2)大鼠急性脊髓損傷后,IL-17、IL-23的表達上調(diào)可能與SCI后繼發(fā)性脊髓損傷的炎癥反應有關。 (3)SCI大鼠急性脊髓損傷后存在STAT3信號轉(zhuǎn)導通路的異常激活,并且可能介導IL-17及其相關細胞因子的分化。(4)在急性脊髓損傷大鼠模型中,可能通過上調(diào)IL-6、IL-21及IL-23等細胞因子的表達促進STAT3信號轉(zhuǎn)導通路的激活,從而介導IL-17細胞因子的表達上調(diào),3者之間可能形成一個循環(huán)相互影響相互促進共同參與急性脊髓損傷后繼發(fā)性脊髓損傷的炎癥反應。
[Abstract]:Spinal cord injury (SCI) is the most serious complication in spinal injury. The latest statistics show that about 250 thousand people in the United States suffer from a different degree of SCI each year, and the annual incidence of 28-50/ is 28-50/. 1 million people, the annual incidence of SCI in Shanghai, China is about 13.7/100 000. And with the development of the world's economic level, the incidence of spinal cord injury is also increasing year by year. There is no definite therapeutic method for the dysfunction caused by spinal cord injury at home and abroad. Therefore, the effective treatment of SCI is studied. There will be great economic and social benefits. Current studies have shown that Th17 cells play a key role in autoimmune mediated tissue damage and organ specific autoimmune reactions. In experimental autoimmune encephalomyelitis (Experimental autoimmune encephalomyelitis, EAE) rats are rich in IL-17 induced CD4+T cells to normal small cells The severity of EAE was induced and the severity of the disease was associated with the level of IL-17. In addition to the changes in the T cell subgroup after the study of SCI, the anti inflammatory cells Th2/Treg and the pro-inflammatory cells Th1/Th17 were activated, suggesting that the Th17 cell effector IL-17A may be involved in the secondary spinal cord injury of SCI. How is the mechanism of IL-17 cytokine immunogenicity in the inflammatory response and how the related cytokine changes and their relationships have not yet been fully elucidated. In order to further elucidate the mechanism of IL-17 cytokine immunogenicity in the inflammatory response to acute spinal cord injury and to seek new therapeutic targets for the treatment of acute spinal cord injury, the issue has been established in this subject. The rat model of spinal cord injury was studied in the following 2 aspects. The first part, IL-17, IL-23 expression in the rat model of acute spinal cord injury,.75 adult healthy male SD rats were randomly divided into control group (15 rats) and group SCI (60),.SCI group was divided into 1H, 24h, 48h and 72h four time subgroups (15 each). The improved Allen's weight was used. The rat model of acute spinal cord injury was successfully established. The model was prepared the day before and after the operation of 1H, 24h, 48h and 72h, and the BBB score was used to detect the pathological changes of the spinal cord after spinal cord injury by.HE staining. The expression of IL-17 in the spinal cord of rats was detected by immunohistochemical staining. Reverse transcription polymerase was used to detect the expression of IL-17 in the spinal cord tissue of rats. The expression of IL-17 and IL-23 in the spleen tissues of each group was detected by the chain reaction (RT-PCR) method.HE staining showed that the morphological structure of the spinal cord was disorder after spinal cord injury, and the white matter and gray matter had obvious signs of compression. The central canal was obviously destroyed, the blood vessel congestion, cell edema, inflammatory cell infiltration, apoptosis and other phenomena in the spinal cord were damaged. .RT-PCR results showed a slight to severe trend in the subgroups of the injured group. Compared with the control group, the expression of IL-17mRNA and IL-23mRNA in the spleen tissue of the spinal cord injury rats increased significantly. The immunohistochemical staining showed that the IL-17 in the spinal cord tissue was almost not expressed in the control group, and the injury to 1H began after the injury. Slightly expressed, the upexpression reached the peak after 24h, and the expression of IL-17 decreased gradually after 48h and 72h. The second part, the differentiation mechanism of IL-17 related cytokines mediated by STAT3 signaling pathway in acute spinal cord injury rats. This part includes two aspects: 1, the expression of STAT3 in the spinal and spleen tissues of rats after spinal cord injury. 2, the serum concentration of related factors regulating IL-17 differentiation in rats after spinal cord injury. In the study of the expression of STAT3 in the spinal cord and spleen tissue of rats after spinal cord injury, the relative expression of p-STAT3 in the spinal cord of the SCI rats was detected by Western blot method, and the spinal cord and spleen group of the spinal cord injury group were compared with the control group. The expression of p-STAT3 was significantly increased in the 24h group, and the expression level of IL-6, IL-21 and IL-23 in the peripheral blood supernatant of SCI rats was detected by ELISA method. The results showed that the serum IL in the spinal cord injury group was compared with the control group. The expression of -6, IL-21 and IL-23 increased significantly in the 24h group. The results of this study suggest that: (1) in SCI rats, with the prolongation of the time of acute spinal cord injury, the range of hemorrhage in the spinal cord is enlarged, the inflammatory response is increased, the number of neuron death is increased, and other pathological changes. (2) after acute spinal cord injury in rats, IL-17, IL-23 The up-regulated expression may be associated with the inflammatory response to secondary spinal cord injury after SCI. (3) there is an abnormal activation of STAT3 signal transduction pathway after acute spinal cord injury in SCI rats, and it may mediate the differentiation of IL-17 and its related cytokines. (4) in the rat model of acute spinal cord injury, it may be promoted by up regulation of IL-6, IL-21 and IL-23. The expression promotes the activation of STAT3 signal transduction pathway, which mediates the up-regulated expression of IL-17 cytokine, which may form a cyclic interaction between the 3 and promoting the joint involvement of the secondary spinal cord injury after acute spinal cord injury.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R651.2

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