应用芬戈莫德调控免疫炎症反应治疗蛛网膜下腔出血的实验研究

发布时间：2018-08-22 17:41

【摘要】：目的自发性蛛网膜下腔出血(SAH)具有高致残率和高死亡率的临床特点。目前,SAH的神经功能障碍尚缺乏行之有效的治疗方法。观察SAH早期免疫炎症反应的变化,并应用新型免疫抑制剂芬戈莫德(Fingolimod)干预,探索SAH治疗的新途径。方法第一部分:于基因表达数据库(GEO)下载小鼠脑基底动脉基因芯片(GSE46696),使用微阵列分析假手术组(Sham组)和模型组(SAH组)样本各3份的基因变化,应用Cytoscape软件对差异表达基因进行功能分析、通路富集分析以及基因-基因互相作用分析。第二部分:假手术组(Sham组)和造模组(SAH组)小鼠各12只,分别于造模后1d、3d各选取6只小鼠应用流式细胞仪检测脑组织自然杀伤细胞(NK)、CD4+和CD25+内源性调节性T细胞(Treg),分别于造模后1d、3d各选取6只小鼠应用Western Blot检测脑组织免疫炎性因子,根据本论文第一部分分析结果,对相对显著的免疫炎性因子或炎症通路IL-1β、TNF-α、NF-κB和TLR4进行检测。第三部分:本部分实验分为假手术组(Sham组)、造模+生理盐水安慰剂组(SAH+Saline组)、造模+芬戈莫德治疗组(SAH+Fingolimod组),SAH+Fingolimod组于造模后2h、48h、72h分别给予Fingolimod(FTY720)腹腔注射,给药浓度为0.1mg/m L(按体重计算每只小鼠给药约200-230μg)。分别于3组中造模给药后第1d、3d抽取小鼠6只,应用流式细胞仪检测模型脑组织自然杀伤细胞(NK)、CD4+和CD25+内源性调节性T细胞;分别于3组中造模给药后第3d抽取小鼠6只,应用Western Blot检测模型脑组织炎症细胞因子或炎症通路IL-1β、TNF-α、NK-κB、TLR4;分别于3组中造模给药后第1d、3d、7d、14d抽取小鼠6只,检测脑组织水含量;分别于3组中造模给药后第1d、3d、7d、14d抽取小鼠6只,自鼠尾静脉注入2%伊文蓝(EB)溶液,检测脑组织EB含量;分别于3组中造模给药后第1d、3d、7d、10d、14d抽取小鼠6只,应用mNNS评分和Morris水迷宫实验,进行小鼠神经功能评估。结果第一部分:小鼠SAH模型脑基底动脉的基因芯片及免疫炎症因子的功能分析,可以发现在SAH早期免疫炎症反应有可能是导致预后不良的重要原因,其中IL-1β、TNF-α等炎症因子和NF-κB、TLR4等炎症通路的显著变化有可能是SAH早期脑损伤(EBI)严重程度的敏感标志之一。第二部分:SAH造模后早期NK细胞/CD3+T细胞的百分比明显增高,与Sham组比较,差异具有统计学意义(均P0.05);SAH组CD4+和Foxp3+T细胞与CD4+T细胞计数的比例,与Sham组比较,略有升高,差异无统计学意义(均P0.05);Western Blot检测脑组织免疫炎性因子或通路IL-1β、TNF-α、NF-κB和TLR4与β-action蛋白的比例明显升高,与Sham组比较,差异具有统计学意义(均P0.05)。第三部分:SAH早期,SAH+Saline组和SAH+Fingolimod组NK细胞、CD3+T细胞计数高于Sham组,SAH+Fingolimod组低于SAH+Saline组,差异具有统计学意义(均P0.05);SAH+Fingolimod组CD4+和Foxp3+T细胞计数高于SAH+Saline组,差异具有统计学意义(均P0.05);SAH+Fingolimod组IL-1β、TNF-α、NF-κB和TLR4表达均低于同时间点SAH组,SAH+Fingolimod组炎性因子或通路的蛋白表达与β-action蛋白的比例均低于同时间点SAH组比较,差异具有统计学意义(均P0.05);第3d、7d检测SAH+Fingolimod组脑组织水含量明显低于SAH+Saline组,差异有统计学意义(均P0.05);第3d、7d检测SAH+Fingolimod组脑组织EB含量明显低于SAH+Saline组,差异有统计学意义(均P0.05);自第3d起SAH+Fingolimod组mNSS评分明显低于SAH+Saline组,差异有统计学意义(P0.05);应用Morris水迷宫评价,SAH+Fingolimod组认知功能改善情况明显好于SAH+Saline组,获得性训练和对位性训练指标比较差异有统计学意义(P0.05)。结论SAH后发生的EBI是多种途径的病理生理变化,对脑组织的损伤非常严重,继而出现的CVS更是加重了脑损伤的程度。免疫炎症反应越来越得到关注与重视,直接影响预后,通过本实验可以初步判定免疫炎症变化是SAH早期脑损伤的重要因素,免疫细胞和炎症因子在脑损伤及修复过程中发挥了重要的作用。这为进一步有针对性的研究和探索SAH早期治疗的药物和方式提供了方向。SAH后出现的免疫炎症反应影响其预后,EBI阶段是治疗干预SAH继发性脑损伤的关键时间段。Fingolimod是一种新型的免疫抑制剂,能够有效的调控免疫功能,降低NK细胞比例水平,升高Treg细胞水平,减轻炎症损伤作用,有助于促进SAH后脑损伤修复,改善BBB通透性,改善SAH预后,这为今后SAH早期药物调控治疗开辟了新的途径,为临床POC研究实施做好基础实验准备,这必将带来前所未有的临床意义和社会效益。SAH早期发生的免疫炎症反应是导致其不良预后的重要原因。新型免疫抑制剂Fingolimod通过调控NK细胞、Treg细胞水平和IL-1β、TNF-α、NF-κB和TLR4等炎症因子或炎症通路变化,干预SAH后EBI期间的病生理反应,缓解CVS的程度,改善SAH预后,为SAH早期药物治疗开辟了新的思路。
[Abstract]:Objective To investigate the clinical characteristics of spontaneous subarachnoid hemorrhage (SAH) with high morbidity and mortality. At present, there is no effective treatment for SAH neurological dysfunction. To observe the changes of early immune inflammation reaction in SAH, and to explore a new way to treat SAH with Fingolimod. Part I: Download mouse basilar artery gene chip (GSE46696) from gene expression database (GEO), analyze the gene changes of sham operation group (Sham group) and model group (SAH group) by microarray, analyze the function of differentially expressed genes, pathway enrichment analysis and gene-gene interaction analysis by Cytoscape software. The second part: 12 mice in sham operation group (Sham group) and 12 mice in SAH group (SAH group), 6 mice in sham operation group (Sham group) and 6 mice in sham operation group (SAH group) were selected to detect the natural killer cells (NK), CD4 + and CD25 + endogenous regulatory T cells (Treg) by flow cytometry on the 1st and 3rd day after modeling, respectively. Six mice in sham operation group (Sham group) and 6 mice in SAH group (SAH group) were selected to detect the brain tissue immunity by Western Blot. Sexual factors, according to the results of the first part of this paper, the relatively significant immunoinflammatory factors or inflammatory pathways IL-1beta, TNF-a, NF-kappa B and TLR4 were detected. Part III: This part of the experiment was divided into sham operation group (Sham group), model + saline placebo group (SAH + Saline group), model + Fengolimod treatment group (SAH + Fingolimod group), SAH + Fingolimod group (SAH + Fingolimod group), SAH + Fingolimod group. The golimod group was injected intraperitoneally with Fingolimod (FTY720) 2 hours, 48 hours and 72 hours after modeling, and the concentration was 0.1 mg/ml (about 200-230 UG per mouse according to body weight). Six mice were selected from the three groups on the 1st and 3rd days after modeling. The endogenous regulatory activity of NK, CD4 + and CD25 + in the brain tissue of the model mice was detected by flow cytometry. T cells were extracted from 6 mice in 3 groups on the 3rd day after modeling administration, and inflammatory cytokines or inflammatory pathways IL-1beta, TNF-alpha, NK-kappa B and TLR4 were detected by Western Blot. Six mice in 3 groups were extracted on the 1st, 3rd, 7th and 14th day after modeling administration, respectively, and the water content in brain tissue was detected. Six mice were injected with 2% Evans blue (EB) solution from the tail vein to detect the content of EB in brain tissue. Six mice were selected from the three groups on the 1st, 3rd, 7th, 10th and 14th day after the injection. The neurological function of the mice was evaluated by mNNS score and Morris water maze test. Functional analysis of these subunits revealed that early immune inflammation may be an important cause of poor prognosis in SAH. Significant changes in inflammatory factors such as IL-1beta, TNF-alpha, and inflammatory pathways such as NF-kappa B and TLR4 may be one of the sensitive markers of the severity of early brain injury (EBI) in SAH. Part II: Early NK cells / CD3 + T after SAH modeling. The percentage of CD4 + and Foxp3 + T cells in SAH group was slightly higher than that in Sham group (all P 0.05). The percentage of CD4 + and Foxp3 + T cells in SAH group was slightly higher than that in Sham group (all P 0.05). There was no significant difference between Western Blot and Sham group in the detection of inflammatory factors or pathways of IL-1 beta, TNF-a, NF-kappa B and TLR4 and beta-actio. Compared with Sham group, the proportion of N protein increased significantly (all P 0.05). Part III: In early SAH, the number of NK cells, CD3 + T cells in SAH + Saline group and SAH + Fingolimod group was higher than Sham group, and the number of CD4 + and Foxp3 + T cells in SAH + Fingolimod group was lower than that in SAH + Saline group (all P 0.05). The expression of IL-1 beta, TNF-a, NF-kappa B and TLR4 in SAH + Fingolimod group was lower than that in SAH + Saline group (all P 0.05), and the expression of inflammatory factors or pathway protein and the ratio of beta-action protein in SAH + Fingolimod group were lower than that in SAH group at the same time point (all P 0.05). The brain water content of SAH + Fingolimod group was significantly lower than that of SAH + Saline group (all P 0.05), and the brain EB content of SAH + Fingolimod group was significantly lower than that of SAH + Saline group on the 3rd and 7th day (all P 0.05), and the mNSS score of SAH + Fingolimod group was significantly lower than that of SAH + Saline group (all P 0.05). Morris water maze test showed that the cognitive function of SAH + Fingolimod group was better than that of SAH + Saline group, and there was significant difference in the indexes of acquired training and contraposition training (P 0.05). Immunoinflammation has attracted more and more attention and has a direct impact on the prognosis. Through this experiment, we can preliminarily determine that immunoinflammation is an important factor of early brain injury in SAH. Immune cells and inflammatory factors play an important role in the process of brain injury and repair. Fingolimod is a new immunosuppressive agent, which can effectively regulate immune function, reduce the proportion of NK cells, and increase Treg cell level. Alleviating inflammation injury is helpful to promote the repair of brain injury after SAH, improve the permeability of BBB, and improve the prognosis of SAH. It opens up a new way for the early drug regulation and treatment of SAH in the future, and provides basic experimental preparation for the implementation of clinical POC research, which will certainly bring unprecedented clinical significance and social benefits. Fingolimod, a new immunosuppressive agent, intervenes in the pathophysiological response of EBI after SAH, alleviates CVS and improves the prognosis of SAH by regulating the levels of NK cells, Treg cells, IL-1beta, TNF-a, NF-kappa B and TLR4.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.35

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