IL-17及其相关细胞因子在大鼠急性脊髓损伤炎症反应中的作用机制研究

发布时间：2018-07-12 20:10

本文选题：IL-17 + STAT3　；参考：《广西医科大学》2015年硕士论文

【摘要】：脊髓损伤(Spinal cord injury, SCI)是指脊髓受到外力的撞击后导致的完全性或不完全性脊髓损伤平面以下的运动、感觉等功能障碍,是脊柱损伤中最严重的并发症。最新的统计数字表明,美国每年约有25万人患不同程度的SCI,年发生率为28-50/100万人,我国上海SCI的年发生率约为13.7/100万人以上。而且随着世界各国经济水平的发展,脊髓损伤发生率也呈现逐年增高的趋势。针对脊髓损伤后导致的功能障碍目前国内外尚无有明确疗效的治疗手段,因此研究有效的治疗SCI的方法将有巨大的经济和社会效益。目前研究表明Th17细胞在自身免疫介导的组织损伤、器官特异自身免疫反应中起着关键的作用。在实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis, EAE)小鼠富含产生IL-17的CD4+T细胞转输给正常小鼠后可诱导出严重的EAE,并且疾病的严重程度与IL-17的水平相关。另外在研究SCI后T细胞亚群的变化时发现,抗炎细胞Th2/Treg和促炎细胞Th1/Th17均有激活,提示Th17细胞的效应分子IL-17A可能参与了SCI的继发性脊髓损伤过程。但在急性脊髓损伤炎症反应中IL-17细胞因子免疫发生机制如何?相关细胞因子变化及其相互关系如何?目前尚未完全阐明。为了进一步阐明急性脊髓损伤炎症反应中IL-17细胞因子免疫发生机制,为治疗急性脊髓损伤寻求新的治疗靶点,本课题建立急性脊髓损伤大鼠模型,进行以下2方面研究。第一部分,急性脊髓损伤大鼠模型不同时期IL-17、IL-23表达的研究。75只成年健康雄性SD大鼠随机分为对照组(15只)和SCI组(60只)。SCI组分成1h、24h、48h及72h四个时间亚组(各15只)。采用改良Allen's重物打击法成功建立大鼠急性脊髓损伤模型,模型制备前一天及术后1h、24h、48h及72h运用BBB评分检测大鼠后肢运动功能。HE染色法检测观察脊髓损伤后脊髓的病理改变,免疫组织化学染色法检测大鼠脊髓组织中IL-17的表达水平。采用反转录-聚合酶链反应(RT-PCR)法检测各组大鼠脾脏组织中IL-17和IL-23的mRNA表达水平。HE染色示脊髓损伤后脊髓组织形态结构紊乱,打击处白质、灰质有明显的压缩迹象,中央管被明显破坏,毛线血管充血、细胞水肿、炎性细胞浸润、细胞凋亡等现象在脊髓损伤各个时间亚组呈现出由轻微至严重的趋势。RT-PCR结果发现：与对照组大鼠相比,脊髓损伤组大鼠脾组织中IL-17mRNA、IL-23mRNA表达量显著上升,具有统计学意义。免疫组织化学染色发现脊髓组织中IL-17在对照组中几乎不表达,损伤1h后开始稍微表达,24h后上升表达达到峰值,损伤48h、72h后IL-17的表达逐渐下降。第二部分,急性脊髓损伤大鼠STAT3信号途径介导的IL-17相关细胞因子的分化机制研究。本部分包括两个方面的研究：1、脊髓损伤后大鼠脊髓和脾脏组织STAT3的表达研究；2、大鼠脊髓损伤后调控大鼠IL-17分化的相关因子的血清浓度鉴定。在脊髓损伤后大鼠脊髓和脾脏组织STAT3的表达研究中,采用Western blot法检测SCI大鼠脊髓、脾脏组织中的p-STAT3的相对表达量,与对照组比较脊髓损伤组大鼠脊髓、脾脏组织中p-STAT3的表达量显著升高以24h组上升最为明显。大鼠脊髓损伤后调控大鼠IL-17分化的相关因子的血清浓度鉴定中,采用ELISA法检测SCI大鼠外周血上清液中的IL-6、IL-21及IL-23的表达水平。结果发现,与对照组比较脊髓损伤组大鼠血清中IL-6、 IL-21及IL-23的表达量显著上升以24h组上升最为明显。本课题研究结果提示：(1)SCI大鼠随着急性脊髓损伤时间的延长脊髓组织呈现出出血范围扩增、炎症反应加剧、神经元死亡数量增加等病理改变。(2)大鼠急性脊髓损伤后,IL-17、IL-23的表达上调可能与SCI后继发性脊髓损伤的炎症反应有关。 (3)SCI大鼠急性脊髓损伤后存在STAT3信号转导通路的异常激活,并且可能介导IL-17及其相关细胞因子的分化。(4)在急性脊髓损伤大鼠模型中,可能通过上调IL-6、IL-21及IL-23等细胞因子的表达促进STAT3信号转导通路的激活,从而介导IL-17细胞因子的表达上调,3者之间可能形成一个循环相互影响相互促进共同参与急性脊髓损伤后继发性脊髓损伤的炎症反应。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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