Malibatol A和重组T细胞受体配体对小鼠大脑中动脉闭塞损伤的脑保护作用及机制研究
发布时间：2018-12-21 13:07
【摘要】：目的缺血性卒中是中国致死率最高的疾病,目前尚缺乏安全有效的治疗方法。免疫炎症反应在脑缺血的疾病过程中具有重要的影响。免疫系统包括外周和中枢,该过程包括多种免疫细胞的活化,如小胶质细胞,巨噬细胞,淋巴细胞,中性粒细胞等。调节小胶质细胞的活化状态,抑制T细胞的病理性激活,对MCAO都具有保护作用。本实验旨在小鼠MCAO模型中探索缺血性卒中的药物治疗方法和机制,包括以下三方面：1、天然白藜芦醇多聚体MalibatolA (MA)保护缺血性卒中的机制研究；2、重组T细胞受体配体(RTL)保护雌性小鼠的MCAO损伤的行为学研究；3、探索一种以啮齿类动物为模型的研究卒中后失语的实验方法。方法利用72小时MCAO脑片的免疫荧光观察MA对M1/M2型小胶质细胞标志物(CD16/32和CD206)在Iba-1+小胶质细胞的表达情况；利用PPARγ抑制剂T0070907后,TTC及NSS检测MA对MCAO梗死体积和神经功能的影响,并利用定量PCR检测对M1/M2型小胶质细胞标志物表型的影响；利用凝胶迁移电泳实验(EMSA)检测MA对PPARγ的转录活性的影响；利用染色质免疫共沉淀(ChIP)检测PPAR与YM-1、CD206启动子序列的结合情况；利用免疫荧光和蛋白质免疫共沉淀检测PPARγ和sumo的共表达情况。利用两种结构的RTL(RTL1000, HLA-DRαl-MOG35-55)在雌性DR2转基因小鼠MCAO后3、24、48、72小时分四次给药,96小时TTC检测其脑梗死体积；利用为期16天的行为学实验,包括cylinder实验、tube turn实验、corner turn实验和novel odor recognition检测RTL治疗后的行为学改变。利用超声检测和分析技术,观察小鼠MCAO后的USV变化并探索RTL治疗对其的影响,并利用PCR和免疫荧光技术探讨其可能的分子机制。结果免疫荧光结果显示MCAO后小胶质细胞激活,MA处理后在Iba-1+细胞中CD16/32表达降低,而CD206增加。T0070907抑制了Malibatol A对MCAO梗死体积的减小作用和对神经功能评分的改善作用。T0070907抑制了MCAO梗死侧皮层MA对M1型标志物(CD16,CD32)的降低作用和对M2型标志物(CD206,YM-1)的增加作用。EMSA和ChIP结果显示,MA增强了PPAR的转录活性,并促进了PPARγ与YM-1启动子序列的结合,但对CD206无明显影响。免疫荧光表明,PPARγ与sumo共表达,但是在MCAO后表达降低。免疫共沉淀结果表明MA能够促进MCAO后PPARγ与sumo的结合。RTL能够降低雌性MCAO梗死体积,其中HLA-DRα1-MOG35-55对梗死体积的影响呈剂量依赖性,其有效剂量高于前期实验中发现的对雄性小鼠的有效剂量。Cylinder实验结果表明RTL1000能够特异性的改善MCAO造成的前肢功能损伤。Novel odor recognition结果在RTL1000治疗及未治疗未见明显差别。同时,本实验建立了一种研究雌性和雄性小鼠MCAO后USV的实验方法,并发现,MCAO后USV的数量降低,频率的复杂度降低。RTL1000对MCAO后USV的数量和频率有影响。MCAO后Foxp2表达降低,可能与USV改变有关。结论MA对MCAO的脑保护和炎症抑制作用与激活PPARγ有关,能够增强PPARγ转录活性,进而上调M2型小胶质细胞的表达。MA的抑制炎症作用可能也与PPARγ的sumo化有关。RTL1000与HLA-DRα 1-MOG-35-55都能够显著的降低雌性小鼠MCAO梗死体积；RTL1000能够特异性的改善MCAO后小鼠运动功能,并对MCAO后USV的数量和频率都有影响。
[Abstract]:Objective Ischemic stroke is the most serious disease in China, and there is still a lack of safe and effective methods for the treatment of ischemic stroke. The immune inflammatory reaction plays an important role in the course of the disease of cerebral ischemia. The immune system includes an outer periphery and a hub, the process comprising activation of a variety of immune cells, such as microglia, macrophages, lymphocytes, neutrophils, and the like. regulating the activation state of the microglia, inhibiting the pathological activation of the T cells, and protecting the MCAO. The purpose of this study is to explore the methods and mechanisms for the treatment of ischemic stroke in the MCAO model of the mouse, including the following three aspects: 1. the study of the mechanism of the protection of ischemic stroke by a natural white-and-white aloe-polymer Maliatroa (MA); 2. A study on the behavior of recombinant T-cell receptor ligand (RTL) in the protection of MCAO injury in female mice. Methods The expression of M1/ M2 microglial cell markers (CD16/ 32 and CD206) in the Iba-1 + microglia was observed by the immunofluorescence of 72-hour MCAO brain slices, and the effects of MA on the volume of MCAO and the neurological function were detected by TTC and NSS after using the PPAR inhibitor T0070907. The effect of MA on the phenotype of M1/ M2 microglial cell marker was detected by quantitative PCR. The effect of MA on the transcription activity of PPAR was detected by gel-transfer electrophoresis (EMSA), and the binding of PPAR with YM-1 and CD206 promoter was detected by means of chromatin immunoprecipitation (ChIP). The co-expression of PPAR antigen and sumo was detected by immunofluorescence and protein immune co-precipitation. Two structures of RTL (RTL1000, HLA-DR-l-MOG35-55) were administered four times at 3, 24, 48, 72 hours after MCAO in female DR2 transgenic mice, and the volume of cerebral infarction was detected by TTC at 96 h; and the behavior change after RTL treatment was detected using a 16-day behavioral experiment, including a cyclinder experiment, a tube return experiment, a corner return experiment, and a novoor recovery test. The changes of the USV and the effect of RTL on the mouse MCAO were observed by means of ultrasonic detection and analysis, and the possible molecular mechanism was discussed by means of PCR and immunofluorescence. Results The results showed that the expression of CD16/ 32 in Iba-1 + cells decreased with the increase of CD206 after MA treatment. T0070907 inhibited the effect of Malibucol A on the volume of MCAO infarction and the improvement of the neurological score. T0070907 inhibited the decrease of the MCAO infarct-side cortical MA on the M1-type marker (CD16, CD32) and the increase in the M2-type marker (CD206, YM-1). The results of EMSA and ChIP show that MA enhances the transcriptional activity of PPAR and promotes the combination of PPAR and YM-1 promoter sequences, but has no significant effect on CD206. Immunofluorescence showed that PPAR was co-expressed with sumo, but decreased after MCAO. The results show that MA can promote the combination of PPAR and sumo after MCAO. The effect of HLA-DR-1-MOG35-55 on the infarct volume was dose-dependent, and the effective dose of HLA-DR-1-MOG35-55 was higher than that in the male mice. The Cyclinder test results show that the RTL1000 can specifically improve the functional damage of the forelimb caused by the MCAO. No significant difference was seen in the treatment and non-treatment of the RL1000. At the same time, this experiment set up a method to study the USV after MCAO in female and male mice, and found that the number of USV after MCAO is reduced and the complexity of frequency is reduced. The RTL1000 has an impact on the number and frequency of the USV after MCAO. The decrease of Foxp2 expression after MCAO may be related to the change of USV. Conclusion The inhibitory effect of MA on the brain protection and inflammation of MCAO is related to the activation of PPAR, which can enhance the transcription activity of PPAR, and then up-regulate the expression of M2-type microglia. The inhibitory effect of MA may also be related to the sumo-ization of PPAR. Both RTL1000 and HLA-DR-1-MOG-35-55 can significantly reduce the MCAO infarct volume of female mice; the RTL1000 can specifically improve the mouse motion function after MCAO, and has an effect on the number and frequency of the USV after MCAO.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3
[Abstract]:Objective Ischemic stroke is the most serious disease in China, and there is still a lack of safe and effective methods for the treatment of ischemic stroke. The immune inflammatory reaction plays an important role in the course of the disease of cerebral ischemia. The immune system includes an outer periphery and a hub, the process comprising activation of a variety of immune cells, such as microglia, macrophages, lymphocytes, neutrophils, and the like. regulating the activation state of the microglia, inhibiting the pathological activation of the T cells, and protecting the MCAO. The purpose of this study is to explore the methods and mechanisms for the treatment of ischemic stroke in the MCAO model of the mouse, including the following three aspects: 1. the study of the mechanism of the protection of ischemic stroke by a natural white-and-white aloe-polymer Maliatroa (MA); 2. A study on the behavior of recombinant T-cell receptor ligand (RTL) in the protection of MCAO injury in female mice. Methods The expression of M1/ M2 microglial cell markers (CD16/ 32 and CD206) in the Iba-1 + microglia was observed by the immunofluorescence of 72-hour MCAO brain slices, and the effects of MA on the volume of MCAO and the neurological function were detected by TTC and NSS after using the PPAR inhibitor T0070907. The effect of MA on the phenotype of M1/ M2 microglial cell marker was detected by quantitative PCR. The effect of MA on the transcription activity of PPAR was detected by gel-transfer electrophoresis (EMSA), and the binding of PPAR with YM-1 and CD206 promoter was detected by means of chromatin immunoprecipitation (ChIP). The co-expression of PPAR antigen and sumo was detected by immunofluorescence and protein immune co-precipitation. Two structures of RTL (RTL1000, HLA-DR-l-MOG35-55) were administered four times at 3, 24, 48, 72 hours after MCAO in female DR2 transgenic mice, and the volume of cerebral infarction was detected by TTC at 96 h; and the behavior change after RTL treatment was detected using a 16-day behavioral experiment, including a cyclinder experiment, a tube return experiment, a corner return experiment, and a novoor recovery test. The changes of the USV and the effect of RTL on the mouse MCAO were observed by means of ultrasonic detection and analysis, and the possible molecular mechanism was discussed by means of PCR and immunofluorescence. Results The results showed that the expression of CD16/ 32 in Iba-1 + cells decreased with the increase of CD206 after MA treatment. T0070907 inhibited the effect of Malibucol A on the volume of MCAO infarction and the improvement of the neurological score. T0070907 inhibited the decrease of the MCAO infarct-side cortical MA on the M1-type marker (CD16, CD32) and the increase in the M2-type marker (CD206, YM-1). The results of EMSA and ChIP show that MA enhances the transcriptional activity of PPAR and promotes the combination of PPAR and YM-1 promoter sequences, but has no significant effect on CD206. Immunofluorescence showed that PPAR was co-expressed with sumo, but decreased after MCAO. The results show that MA can promote the combination of PPAR and sumo after MCAO. The effect of HLA-DR-1-MOG35-55 on the infarct volume was dose-dependent, and the effective dose of HLA-DR-1-MOG35-55 was higher than that in the male mice. The Cyclinder test results show that the RTL1000 can specifically improve the functional damage of the forelimb caused by the MCAO. No significant difference was seen in the treatment and non-treatment of the RL1000. At the same time, this experiment set up a method to study the USV after MCAO in female and male mice, and found that the number of USV after MCAO is reduced and the complexity of frequency is reduced. The RTL1000 has an impact on the number and frequency of the USV after MCAO. The decrease of Foxp2 expression after MCAO may be related to the change of USV. Conclusion The inhibitory effect of MA on the brain protection and inflammation of MCAO is related to the activation of PPAR, which can enhance the transcription activity of PPAR, and then up-regulate the expression of M2-type microglia. The inhibitory effect of MA may also be related to the sumo-ization of PPAR. Both RTL1000 and HLA-DR-1-MOG-35-55 can significantly reduce the MCAO infarct volume of female mice; the RTL1000 can specifically improve the mouse motion function after MCAO, and has an effect on the number and frequency of the USV after MCAO.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R743.3
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